Dihydropyrancarboxamides and uses thereof

ABSTRACT

The present invention provides novel dihydropyrancarboxamide compounds of formula (I):  
                 
         and collections of these compounds, and provides methods for the synthesis of these compounds; wherein R 1 -R 6  are as defined herein. Additionally, the present invention provides pharmaceutical compositions and methods for treating disorders such as proliferative diseases, and cancer, to name a few.

PRIORITY INFORMATION

The present application claims priority under 35 U.S.C. § 119 to U.S.provisional application No. 60/406,140, filed Aug. 27, 2002, entitled“Dihydropyrancarboxamides and Uses Thereof”, the entire contents ofwhich are hereby incorporated by reference.

GOVERNMENT SUPPORT

This invention was made in part with a grant from the National Instituteof General Medical Sciences (Grant Number: GM-52067). Therefore, thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

The identification of small organic molecules that affect specificbiological functions is an endeavor that impacts both biology andmedicine. Such molecules are useful as therapeutic agents and as probesof biological function. In but one example from the emerging field ofchemical genetics, in which small molecules are used to alter thefunction of biological molecules to which they bind, small moleculeshave helped elucidate signal transduction pathways by acting as chemicalprotein knockouts. (Schreiber et al., J. Am. Chem. Soc., 1990, 112,5583; Mitchison, Chem. and Biol., 1994, 1, 3). Of course, smallmolecules that interact with particular biological targets and affectspecific biological functions, may also serve as candidates for thedevelopment of therapeutics. One important class of small molecules arenatural products, which are small molecules obtained from nature.Natural products have played an important role in the development ofbiology and medicine, serving as pharmaceutical leads, drugs (Newman etal., Nat. Prod. Rep. 2000, 17, 215-234), and powerful reagents forstudying cell biology (Schreiber, S. L. Chem. and Eng News 1992 (October26), 22-32). More generally, any organic compounds, whethernaturally-occurring, reminiscent of natural products or artificiallycreated (e.g., via chemical synthesis or semi-synthesis), are also ofinterest since they may serve as candidates for the development oftherapeutics.

Because it is difficult to predict which small molecules will interactwith a biological target, and it is often difficult to obtain orefficiently synthesize small molecules found in nature, intense effortshave been directed toward the generation of large numbers, or libraries,of small organic compounds, often “natural product-like” libraries.These libraries can be tested in sensitive assays for a particularbiological activity, such as binding to a target of interest.

Clearly, it would be desirable to develop compounds with a desiredbiological activity. Additionally, it would be desirable to identifynovel compounds capable of acting as probes of biological function.

SUMMARY OF THE INVENTION

In one aspect of the invention, novel compounds having the structure (1)are provided:

wherein R¹-R⁴ are each independently hydrogen or an aliphatic,heteroaliphatic, aryl, heteroaryl, alkylaryl or alkylheteroaryl moiety;

R⁵ and R⁶ are each independently hydrogen or an aliphatic,heteroaliphatic, aryl, heteroaryl, alkylaryl, or alkylheteroaryl moiety,and wherein R⁵ and R⁶, taken together, may form a cyclic aliphatic,heteroaliphatic, aliphatic(aryl), heteroaliphatic(aryl),aliphatic(heteroaryl) or heteroaliphatic(heteroaryl) moiety, or an arylor heteroaryl moiety;

wherein each of the foregoing aliphatic and heteroaliphatic moieties maybe substituted or unsubstituted, cyclic or acyclic, saturated orunsaturated or linear or branched; and each of the foregoing aryl,heteroaryl, alkylaryl or alkylheteroaryl moieties may be substituted orunsubstituted; and

pharmaceutically acceptable derivatives thereof.

In certain embodiments, compounds having the structure (II) areprovided:

or enantiomer thereof;

wherein R¹-R⁴ are each independently hydrogen or an aliphatic,heteroaliphatic, aryl, heteroaryl, alkylaryl or alkylheteroaryl moiety;

R⁵ and R⁶ are each independently hydrogen or an aliphatic,heteroaliphatic, aryl, heteroaryl, alkylaryl, or alkylheteroaryl moiety,and wherein R⁵ and R⁶, taken together, may form a cyclic aliphatic,heteroaliphatic, aliphatic(aryl), heteroaliphatic(aryl),aliphatic(heteroaryl) or heteroaliphatic(heteroaryl) moiety, or an arylor heteroaryl moiety;

wherein each of the foregoing aliphatic and heteroaliphatic moieties maybe substituted or unsubstituted, cyclic or acyclic, saturated orunsaturated or linear or branched; and each of the foregoing aryl,heteroaryl, alkylaryl or alkylheteroaryl moieties may be substituted orunsubstituted; and

pharmaceutically acceptable derivatives thereof.

In yet other embodiments, a collection of compounds comprising two ormore of the compounds of structures (I) or (II) is provided. In certainembodiments, the collection is provided in array format. In yet otherembodiments, the collection is provided in array format on a glassslide. In still other embodiments, the collection comprises at least 100compounds. In yet other embodiments, the collection comprises at least1,000 compounds. In still further embodiments, the collection comprisesat least 2,000 compounds. In yet other embodiments, the collectioncomprises at least 10,000 compounds.

In another aspect of the invention, a method for the synthesis of thecore structure (III) is provided, one method comprising steps of:

providing a vinyl ether having the structure:

providing an unsaturated ketoester having the structure:

subjecting the vinyl ether and the unsaturated ketoester to suitableconditions to generate a scaffold having the core structure:

wherein R¹ and R² are each independently hydrogen or an aliphatic,heteroaliphatic, aryl, heteroaryl, alkylaryl or alkylheteroaryl moiety;wherein one of R¹ or R² is attached to a solid support;

R³ and R⁴ are each independently hydrogen or an aliphatic,heteroaliphatic, aryl, heteroaryl, alkylaryl or alkylheteroaryl moiety;

R^(A) is hydrogen or is an aliphatic, heteroaliphatic, aryl, heteroaryl,alkylaryl, or alkylheteroaryl moiety;

wherein each of the foregoing aliphatic and heteroaliphatic moieties maybe substituted or unsubstituted, cyclic or acyclic, saturated orunsaturated or linear or branched; and each of the foregoing aryl,heteroaryl, alkylaryl or alkylheteroaryl moieties may be substituted orunsubstituted.

In certain embodiments, the method further comprises cleaving the corestructure (III) from the solid support to which it is attached.

In certain embodiments, the method further comprises subjecting the corestructure (III) to one or more diversification reactions to generate oneor more compounds having the structure (I):

wherein R¹ and R⁶ are as defined above.

In certain embodiments, the method further comprises cleaving the corestructure (I) from the solid support to which it is attached.

In yet another aspect of the invention, pharmaceutical compositions areprovided comprising any one of the compounds described above and herein;and a pharmaceutically acceptable carrier or diluent.

In still another aspect of the invention, methods of treating a varietyof disorders are provided comprising administering a therapeuticallyeffective compound or composition thereof to a subject in need thereof.In certain other embodiments, the inventive compounds are utilized totreat proliferative disorders, including, but not limited to cancer.

In yet another aspect of the present invention, methods of screeningcompounds for identifying those inventive compounds that exhibit abiological activity of interest are provided.

DEFINITIONS

This invention provides a new family of compounds with a range ofbiological properties. Compounds of this invention have biologicalactivities relevant for the treatment of diseases includingproliferative diseases such as cancer. Compounds of this inventioninclude those specifically set forth above and described herein, and areillustrated in part by the various classes, subgenera and speciesdisclosed elsewhere herein.

It will be appreciated by one of ordinary skill in the art thatasymmetric centers may exist in the compounds of the present invention.Thus, inventive compounds and pharmaceutical compositions thereof may bein the form of an individual enantiomer, diastereomer or geometricisomer, or may be in the form of a mixture of stereoisomers. In certainembodiments, the compounds of the invention are enantiopure compounds.In certain other embodiments, a mixtures of stereoisomers ordiastereomers are provided.

Additionally, the present invention provides pharmaceutically acceptablederivatives of the inventive compounds, and methods of treating asubject using these compounds, pharmaceutical compositions thereof, oreither of these in combination with one or more additional therapeuticagents. The phrase, “pharmaceutically acceptable derivative”, as usedherein, denotes any pharmaceutically acceptable salt, ester, or salt ofsuch ester, of such compound, or any other adduct or derivative which,upon administration to a patient, is capable of providing (directly orindirectly) a compound as otherwise described herein, or a metabolite orresidue thereof. Pharmaceutically acceptable derivatives thus includeamong others pro-drugs. A pro-drug is a derivative of a compound,usually with significantly reduced pharmacological activity, whichcontains an additional moiety which is susceptible to removal in vivoyielding the parent molecule as the pharmacologically active species. Anexample of a pro-drug is an ester which is cleaved in vivo to yield acompound of interest. Pro-drugs of a variety of compounds, and materialsand methods for derivatizing the parent compounds to create thepro-drugs, are known and may be adapted to the present invention.Certain exemplary pharmaceutical compositions and pharmaceuticallyacceptable derivatives will be discussed in more detail below.

Certain compounds of the present invention, and definitions of specificfunctional groups are also described in more detail below. For purposesof this invention, the chemical elements are identified in accordancewith the Periodic Table of the Elements, CAS version, Handbook ofChemistry and Physics, 75th Ed., inside cover, and specific functionalgroups are generally defined as described therein. Additionally, generalprinciples of organic chemistry, as well as specific functional moietiesand reactivity are described in “Organic Chemistry”, Thomas Sorrell,University Science Books, Sausalito: 1999, the entire contents of whichare incorporated herein by reference. Furthermore, it will beappreciated by one of ordinary skill in the art that the syntheticmethods, as described herein, may utilize a variety of protectinggroups. By the term “protecting group”, has used herein, it is meantthat a particular functional moiety, e.g., O, S, or N, is temporarilyblocked so that a reaction can be carried out selectively at anotherreactive site in a multifunctional compound. In preferred embodiments, aprotecting group reacts selectively in good yield to give a protectedsubstrate that is stable to the projected reactions; the protectinggroup should be selectively removed in good yield by readily available,preferably nontoxic reagents that do not attack the other functionalgroups; the protecting group forms an easily separable derivative (morepreferably without the generation of new stereogenic centers); and theprotecting group has a minimum of additional functionality to avoidfurther sites of reaction. As detailed herein, oxygen, sulfur, nitrogenand carbon protecting groups may be utilized. Exemplary protectinggroups are detailed herein, however, it will be appreciated that thepresent invention is not intended to be limited to these protectinggroups; rather, a variety of additional equivalent protecting groups canbe readily identified using the above criteria and utilized in themethod of the present invention. Additionally, a variety of protectinggroups are described in “Protective Groups in Organic Synthesis” ThirdEd. Greene, T. W. and Wuts, P. G., Eds., John Wiley & Sons, New York:1999, the entire contents of which are hereby incorporated by reference.

It will be appreciated that the compounds described herein, may besubstituted with any number of substituents or functional moieties. Ingeneral, the term “substituted” whether preceded by the term“optionally” or not, and substituents contained in formulas of thisinvention, refer to the replacement of hydrogen radicals in a givenstructure with the radical of a specified substituent. When more thanone position in any given structure may be substituted with more thanone substituent selected from a specified group, the substituent may beeither the same or different at every position unless otherwiseindicated. As used herein, the term “substituted” is contemplated toinclude all permissible substituents of organic compounds. In a broadaspect, the permissible substituents include acyclic and cyclic,branched and unbranched, carbocyclic and heterocyclic, aromatic andnonaromatic substituents of organic compounds. For purposes of thisinvention, heteroatoms such as nitrogen may have hydrogen substituentsand/or any permissible substituents of organic compounds describedherein which satisfy the valencies of the heteroatoms. Furthermore, thisinvention is not intended to be limited in any manner by the permissiblesubstituents of organic compounds. Combinations of substituents andvariables envisioned by this invention are preferably those that resultin the formation of stable compounds useful in the treatment, forexample, of proliferative disorders, cancer, and wound healing, to namea few. The term “stable”, as used herein, preferably refers to compoundswhich possess stability sufficient to allow manufacture and whichmaintain the integrity of the compound for a sufficient period of timeto be detected and preferably for a sufficient period of time to beuseful for the purposes detailed herein.

The term “aliphatic”, as used herein, includes both saturated andunsaturated, straight chain (i.e., unbranched), branched, cyclic, orpolycyclic aliphatic hydrocarbons, which are optionally substituted withone or more functional groups. As will be appreciated by one of ordinaryskill in the art, “aliphatic” is intended herein to include, but is notlimited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, andcycloalkynyl moieties. Thus, as used herein, the term “alkyl” includesboth straight, branched and cyclic alkyl groups. An analogous conventionapplies to other generic terms such as “alkenyl”, “alkynyl” and thelike. Furthermore, as used herein, the terms “alkyl”, “alkenyl”,“alkynyl” and the like encompass both substituted and unsubstitutedgroups.

In certain embodiments, the alkyl, alkenyl and alkynyl groups employedin the invention contain 1-20 aliphatic carbon atoms. In certain otherembodiments, the alkyl, alkenyl, and alkynyl groups employed in theinvention contain 1-10 aliphatic carbon atoms. In still otherembodiments, the alkyl, alkenyl, and alkynyl groups employed in theinvention contain 1-6 aliphatic carbon atoms. In yet other embodiments,the alkyl, alkenyl, and alkynyl groups employed in the invention contain1-4 aliphatic carbon atoms. Illustrative aliphatic groups thus include,but are not limited to, for example, methyl, ethyl, n-propyl, isopropyl,cyclopropyl, —CH₂-cyclopropyl, allyl, n-butyl, sec-butyl, isobutyl,tert-butyl, cyclobutyl, —CH₂-cyclobutyl, n-pentyl, sec-pentyl,isopentyl, tert-pentyl, cyclopentyl, —CH₂-cyclopentyl, n-hexyl,sec-hexyl, cyclohexyl, —CH₂-cyclohexyl moieties and the like, whichagain, may bear one or more substituents. Alkenyl groups include, butare not limited to, for example, ethenyl, propenyl, butenyl,1-methyl-2-buten-1-yl, and the like. Representative alkynyl groupsinclude, but are not limited to, ethynyl, 2-propynyl(propargyl),1-propynyl and the like.

The term “alkoxy”, or “thioalkyl” as used herein refers to an alkylgroup, as previously defined, attached to the parent molecular moietythrough an oxygen atom or through a sulfur atom. In certain embodiments,the alkyl group contains 1-20 alipahtic carbon atoms. In certain otherembodiments, the alkyl group contains 1-10 aliphatic carbon atoms. Instill other embodiments, the alkyl group contains 1-6 aliphatic carbonatoms. In yet other embodiments, the alkyl group contains 1-4 aliphaticcarbon atoms. Examples of alkoxy, include but are not limited to,methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, neopentoxyand n-hexoxy. Examples of thioalkyl include, but are not limited to,methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, and thelike.

The term “alkylamino” refers to a group having the structure —NHR′wherein R′ is alkyl, as defined herein. In certain embodiments, thealkyl group contains 1-20 aliphatic carbon atoms. In certain otherembodiments, the alkyl group contains 1-10 aliphatic carbon atoms. Instill other embodiments, the alkyl group contains 1-6 aliphatic carbonatoms. In yet other embodiments, the alkyl group contains 1-4 aliphaticcarbon atoms. Examples of alkylamino include, but are not limited to,methylamino, ethylamino, iso-propylamino and the like.

Some examples of substituents of the above-described aliphatic (andother) moieties of compounds of the invention include, but are notlimited to aliphatic; heteroaliphatic; aryl; heteroaryl; alkylaryl;alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy;alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br; I; —OH;—NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂;—CH₂SO₂CH₃; —C(O)R_(x); —CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x);—OCO₂R_(x); —OCON(R_(x))₂; —N(R_(x))₂; —S(O)₂R_(x); wherein eachoccurrence of R_(x) independently includes, but is not limited to,aliphatic, heteroaliphatic, aryl, heteroaryl, alkylaryl, oralkylheteroaryl, wherein any of the aliphatic, heteroaliphatic,alkylaryl, or alkylheteroaryl substituents described above and hereinmay be substituted or unsubstituted, branched or unbranched, cyclic oracyclic, and wherein any of the aryl or heteroaryl substituentsdescribed above and herein may be substituted or unsubstituted.Additional examples of generally applicable substituents are illustratedby the specific embodiments shown in the Examples which are describedherein.

In general, the terms “aryl” and “heteroaryl”, as used herein, refer tostable mono- or polycyclic, heterocyclic, polycyclic, andpolyheterocyclic unsaturated moieties having preferably 3-14 carbonatoms, each of which may be substituted or unsubstituted. Substituentsinclude, but are not limited to, any of the previously mentionedsubstitutents, i.e., the substituents recited for aliphatic moieties, orfor other moieties as disclosed herein, resulting in the formation of astable compound. In certain embodiments of the present invention, “aryl”refers to a mono- or bicyclic carbocyclic ring system having one or twoaromatic rings including, but not limited to, phenyl, naphthyl,tetrahydronaphthyl, indanyl, indenyl and the like. In certainembodiments of the present invention, the term “heteroaryl”, as usedherein, refers to a cyclic aromatic radical having from five to ten ringatoms of which one ring atom is selected from S, O and N; zero, one ortwo ring atoms are additional heteroatoms independently selected from S,O and N; and the remaining ring atoms are carbon, the radical beingjoined to the rest of the molecule via any of the ring atoms, such as,for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl,imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl,thiophenyl, furanyl, quinolinyl, isoquinolinyl, and the like.

It will be appreciated that aryl and heteroaryl groups (includingbicyclic aryl groups) can be unsubstituted or substituted, whereinsubstitution includes replacement of one, two or three of the hydrogenatoms thereon independently with any one or more of the followingmoieties including, but not limited to: aliphatic; heteroaliphatic;aryl; heteroaryl; alkylaryl; alkylheteroaryl; alkoxy; aryloxy;heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio;heteroarylthio; F; Cl; Br; I; —OH; —NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂;—CH₂OH; —CH₂CH₂OH; —CH₂NH₂; —CH₂SO₂CH₃; —C(O)R_(x); —CO₂(R_(x));—CON(R_(x))₂; —OC(O)R_(x); —OCO₂R_(x); —OCON(R_(x))₂; —N(R_(x))₂;—S(O)₂R_(x); wherein each occurrence of R_(x) independently includes,but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl,alkylaryl, or alkylheteroaryl, wherein any of the aliphatic,heteroaliphatic, alkylaryl, or alkylheteroaryl substituents describedabove and herein may be substituted or unsubstituted, branched orunbranched, cyclic or acyclic, and wherein any of the aryl or heteroarylsubstituents described above and herein may be substituted orunsubstituted. Additional examples of generally applicable substitutentsare illustrated by the specific embodiments shown in the Examples whichare described herein.

The term “cycloalkyl”, as used herein, refers specifically to groupshaving three to seven, preferably three to ten carbon atoms. Suitablecycloalkyls include, but are not limited to cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl and the like, which, as in the caseof other aliphatic, heteroaliphatic or heterocyclic moieties, mayoptionally be substituted with substituents including, but not limitedto aliphatic; heteroaliphatic; aryl; heteroaryl; alkylaryl;alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy;alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br; I; —OH;—NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂;—CH₂SO₂CH₃; —C(O)R_(x); —CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x);—OCO₂R_(x); —OCON(R_(x))₂; —N(R_(x))₂; —S(O)₂R_(x); wherein eachoccurrence of R_(x) independently includes, but is not limited to,aliphatic, heteroaliphatic, aryl, heteroaryl, alkylaryl, oralkylheteroaryl, wherein any of the aliphatic, heteroaliphatic,alkylaryl, or alkylheteroaryl substituents described above and hereinmay be substituted or unsubstituted, branched or unbranched, cyclic oracyclic, and wherein any of the aryl or heteroaryl substituentsdescribed above and herein may be substituted or unsubstituted.Additional examples of generally applicable substitutents areillustrated by the specific embodiments shown in the Examples which aredescribed herein.

The term “heteroaliphatic”, as used herein, refers to aliphatic moietieswhich contain one or more oxygen, sulfur, nitrogen, phosphorous orsilicon atoms, e.g., in place of carbon atoms. Heteroaliphatic moietiesmay be branched, unbranched or cyclic and include saturated andunsaturated heterocycles such as morpholino, pyrrolidinyl, etc. Incertain embodiments, heteroaliphatic moieties are substituted byindependent replacement of one or more of the hydrogen atoms thereonwith one or more moieties including, but not limited to aliphatic;heteroaliphatic; aryl; heteroaryl; alkylaryl; alkylheteroaryl; alkoxy;aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio;heteroalkylthio; heteroarylthio; F; Cl; Br; I; —OH; —NO₂; —CN; —CF₃;—CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂; —CH₂SO₂CH₃; —C(O)R_(x);—CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x); —OCO₂R_(x); —OCON(R_(x))₂;—N(R_(x))₂; —S(O)₂R_(x); wherein each occurrence of R_(x) independentlyincludes, but is not limited to, aliphatic, heteroaliphatic, aryl,heteroaryl, alkylaryl, or alkylheteroaryl, wherein any of the aliphatic,heteroaliphatic, alkylaryl, or alkylheteroaryl substituents describedabove and herein may be substituted or unsubstituted, branched orunbranched, cyclic or acyclic, and wherein any of the aryl or heteroarylsubstituents described above and herein may be substituted orunsubstituted. Additional examples of generally applicable substitutentsare illustrated by the specific embodiments shown in the Examples whichare described herein.

The terms “halo” and “halogen” as used herein refer to an atom selectedfrom fluorine, chlorine, bromine and iodine.

The term “haloalkyl” denotes an alkyl group, as defined above, havingone, two, or three halogen atoms attached thereto and is exemplified bysuch groups as chloromethyl, bromoethyl, trifluoromethyl, and the like.

The term “heterocycloalkyl” or “heterocycle”, as used herein, refers toa non-aromatic 5-, 6- or 7-membered ring or a polycyclic group,including, but not limited to a bi- or tri-cyclic group comprising fusedsix-membered rings having between one and three heteroatomsindependently selected from oxygen, sulfur and nitrogen, wherein (i)each 5-membered ring has 0 to 1 double bonds and each 6-membered ringhas 0 to 2 double bonds, (ii) the nitrogen and sulfur heteroatoms may beoptionally be oxidized, (iii) the nitrogen heteroatom may optionally bequaternized, and (iv) any of the above heterocyclic rings may be fusedto a benzene ring. Representative heterocycles include, but are notlimited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl,imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl,morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl. Incertain embodiments, a “substituted heterocycloalkyl or heterocycle”group is utilized and as used herein, refers to a heterocycloalkyl orheterocycle group, as defined above, substituted by the independentreplacement of one, two or three of the hydrogen atoms thereon with butare not limited to aliphatic; heteroaliphatic; aryl; heteroaryl;alkylaryl; alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy;heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F;Cl; Br; I; —OH; —NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH;—CH₂NH₂; —CH₂SO₂CH₃; —C(O)R_(x); —CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x);—OCO₂R_(x); —OCON(R_(x))₂; —N(R^(x))₂; —S(O)₂R_(x); wherein eachoccurrence of R_(x) independently includes, but is not limited to,aliphatic, heteroaliphatic, aryl, heteroaryl, alkylaryl, oralkylheteroaryl, wherein any of the aliphatic, heteroaliphatic,alkylaryl, or alkylheteroaryl substituents described above and hereinmay be substituted or unsubstituted, branched or unbranched, cyclic oracyclic, and wherein any of the aryl or heteroaryl substituentsdescribed above and herein may be substituted or unsubstituted.Additional examples of generally applicable substitutents areillustrated by the specific embodiments shown in the Examples which aredescribed herein.

The term “solid support”, as used herein, refers to a material having arigid or semi-rigid surface. Such materials will preferably take theform of small beads, pellets, disks, chips, dishes, multi-well plates,glass slides, wafers, or the like, although other forms may be used. Insome embodiments, at least one surface of the substrate will besubstantially flat. The term “surface” refers to any generallytwo-dimensional structure on a solid substrate and may have steps,ridges, kinks, terraces, and the like without ceasing to be a surface.

The term “polymeric support”, as used herein, refers to a soluble orinsoluble polymer to which an amino acid or other chemical moiety can becovalently bonded by reaction with a functional group of the polymericsupport. Many suitable polymeric supports are known, and include solublepolymers such as polyethylene glycols or polyvinyl alcohols, as well asinsoluble polymers such as polystyrene resins. A suitable polymericsupport includes functional groups such as those described below. Apolymeric support is termed “soluble” if a polymer, or apolymer-supported compound, is soluble under the conditions employed.However, in general, a soluble polymer can be rendered insoluble underdefined conditions. Accordingly, a polymeric support can be solubleunder certain conditions and insoluble under other conditions.

The term “linker”, as used herein, refers to a chemical moiety utilizedto attach a compound of interest to a solid support to facilitatesynthesis of inventive compounds. Exemplary linkers are described inExample 2, as described herein. It will be appreciated that otherlinkers (including silicon-based linkers and other linkers) that areknown in the art can also be employed for the synthesis of the compoundsof the invention.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 depicts an exemplary synthesis of the inventivedihydropyrancarboxamides.

FIG. 2 depicts an exemplary encoded split-pool synthesis ofdihydropyrancarboxamides, with the (S)-1 catalyst. The correspondingopposite enantiomers of compounds 7-15 are obtained when the (R)-1catalyst is used. Encircled R¹ and R² symbols represent elements foundin building blocks BB1-A-H in FIG. 3.PyBOP=benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate,DMF=N,N-dimethylformamide, THF=tetrahydrofuran.

FIG. 3 depicts building blocks for the inventive dihydropyrancarboxamidelibraries.

FIG. 4 depicts four bright-field microscopy images ofsilicon-functionalized polystyrene resins that have been subjected todifferent washing and drying experiments as described in the text: (a)an image of “reference beads”; (b) an image of “gentle conditions”; (c)an image of “best practice” beads; (d) a magnified image of a typicalbroken bead found in the “damaged beads”.

FIG. 5 depicts a graphical representation of the optimization ofdiazoketone tag and rhodium catalyst 5 concentration in encodingreactions.

FIG. 6A depicts a graph of quantitative GC data for encoding test resin6 with Tags C3Cl3, C3Cl5, C9Cl5, and C16Cl5 with various tag pre-soakingtimes. Encoding conditions: 2 h reaction time after addition of Tags orcatalyst 5, 25° C. Decoding conditions: 0.25 M CAN (1:1 THF/H₂O), 2 h,25° C., 1 min sonication, 1 μL N,O-bis(trimethylsilyl)aceteamide (BSA).Each data point is an average of 10 identical experiments withindividual beads.

FIG. 6B depicts a graph of quantitative GC data for a time courseexperiment for encoding test resin 6 with Tags C3Cl3, C3Cl5, C9Cl5, andC16Cl5. Encoding conditions: pre-soak with Tags 45 min prior to additionof catalyst 5, quench reactions by the addition of 5 μL heptylamine.Decoding conditions: 0.24 M CAN (5:1 THF/H₂O), 21 h, 37° C., 1 minsonication, 1 μL 1:1 BSA/decane. Each data point is an average of 10identical experiments with individual beads.

FIG. 6C depicts a graph of quantitative GC data for the decoding onebead of test resin 6 before compound cleavage, after compound cleavage,or from 100% of the cleaved compound. Encoding conditions: 45 min tagpre-soak prior to addition of catalyst 5, 2 h, 25° C. Decodingconditions: 0.25 M CAN (1:1 THF/H₂O), 2 h, 25° C., 1 min sonication, 1μL BSA. Each data point is an average of 10 identical experiments withindividual beads.

FIG. 6D depicts a graph of quantitative GC data for a time courseexperiment for decoding test resin 7. Decoding conditions: 0.25 M CAN(1:1 THF/H₂O), 25° C., 1 min sonication, 1 μL 1:1 BSA:decane. Each datapoint is an average of 10 identical experiments with individual beads.

FIG. 6E depicts a graph of quantitative GC data for decoding test resin7 at various temperatures. Reactions stored at room temperature, placedin a 37° C. incubator, or placed in a 60° C. oven. Decoding conditions:0.25 M CAN (1:1 THF/H₂O), 2 h, 1 min sonication, 1 μL 1:1 BSA:decane.Each data point is an average of 10 identical experiments withindividual beads.

FIG. 6F depicts a graph of quantitative GC data for the decoding of testresin 7 with varying CAN solution concentrations and solventcompositions. Decoding conditions: 21 h, 37° C., 1 min sonication, 1 μL1:1 BSA:decane. Each data point is an average of 10 identicalexperiments with individual beads.

FIG. 6G depicts a graph of quantitative GC data for subjection ofdecoding test resin 7 to various time periods of sonication after CANcleavage. Decoding conditions: 0.25 M CAN (1:1 THF/H₂O), 2 h, 25° C., 1μL 1:1 BSA:decane. Each data point is an average of 10 identicalexperiments with individual beads.

FIG. 6H depicts a graph of quantitative GC data for subjection of Tagalcohols cleaved from decoding test resin 7 to various amounts ofBSA/decane solutions prior to GC analyses. Decoding conditions: 0.24 MCAN (5:1 THF/H₂O), 21 h, 37° C., 1 min sonication. Each data point is anaverage of 10 identical experiments with individual beads.

FIG. 6I depicts a graph of quantitative GC data for the decoding of testresin 7 using either our optimized decoding protocol for 500-600 μmpolystyrene beads or the decoding protocol reported by the PharmacopeiaCompany for 90 μm TentaGel (See Dolle, R. E.; Guo, J.; O'Brien, L.; Jin,Y.; Piznik, M.; Bowman, K. J.; Li, W.; Ehan, W. I.; Carvallaro, C.;Roughton, A. L.; Zhao, Q.; Reader, J. C.; Orlowski, M.; Jacob-Samuel,B.; Carroll, C. D. J. Comb. Chem. 2000, 2, 716-731). Each data point isan average of 10 identical experiments with individual beads.

FIG. 7 depicts purity data determined by LC/MS for the 108representative compounds cleaved from library 12, as described inExample 2 herein.

FIGS. 8A-8D depicts structures of the 54 compounds cleaved from beadschosen from batches of resin exposed to the S—Cu (II) catalyst inlibrary 12, as described in Example 2 herein. Numbers in bold refer tobead number. Except for compound 105, all structures showed agreementbetween GC decoding and MS data.

FIGS. 9A and 9B depict representative examples of GC (a, b), LC (c, d),and MS (e, f) spectra from bead and stock-solution decoding (samples 12and 48, respectively), as described in Example 2 herein. Thebead-decoding GC trace for sample 12 (a) decodes for a library compoundwith an exact mass identical to that obtained by MS (e) of the compoundcleaved from that bead (APCI, observed mass=479.9 [M+1]). Thestock-solution-decoding GC trace for sample 48 (b) decodes for a librarycompound with an exact mass identical to that obtained by MS (f) of thecompound stock solution (APCI, observed mass=626.8 [M+1]). The singlepeaks in the LC spectra (c, d) correspond to these molecular ions. [Thestarred peak (*) in the GC traces (a, b) is an impurity frequentlypresent with the electrophoric tags.]

FIG. 10 outlines an exemplary embodiment of the invention: a ‘one-bead,one-stock solution’ technology platform directed toward chemicalgenetics. DVB=divinylbenzene.

FIG. 11 depicts an overview of exemplary library formatting andannotation screening.

FIG. 12 depicts a bead arrayer. The bead arrayer is (a) attached tostandard nitrogen and vacuum lines. Vacuum is applied and beads are (b)decanted onto the platform for entrainment by 384 depressions. Excessbeads are recovered, leaving (c) a regular array of 384 beads withidentical spacing to a standard 384-well microtiter plate.

FIG. 13 depicts a representative reverse chemical genetic assay, asdescribed in Example 3 herein. A small molecule microarray containingmembers of 10 was probed with purified Cy5-labeled (His)6-FKBP12. a:Fluorescence intensity at duplicate spots (false-colored red) containinga ‘hit’ is shown compared to a rhodamine control spot (false-coloredgreen). b: The structure of the ‘hit’ (11) was determined by beaddecoding and confirmed by LC/MS.

FIG. 14 depicts representative forward chemical genetic assays asdescribed in Example 3 herein. Human A549 cells were exposed induplicate to stock solutions of 10. ‘Hits’ (gray boxes) are those wellsthat scored in both replicates of a given experiment. Data from 48representative wells are shown as multiplicative overlays of cytoblotresults from (a) a BrdU incorporation assay, and (b) a genisteinsuppressor screen. c: Structures of representative ‘hits’. Beads orstock solutions corresponding to ‘hits’ in cytoblot assays were exposedto the optimized decoding protocol described in the preceding paper inthis issue. Compounds are labeled by well position in the assay plates.

FIG. 15 depicts an exemplary inventive compound identified in an Eg5inhibitor assay, as described in Example 3 herein.

FIGS. 16A-16D depict LC traces from 25 inventive livrary members (e.g.,quality control compounds).

FIGS. 17A-17C depict MS traces corresponding to the LC traces of FIGS.16A-16D.

FIG. 18 depicts exemplary raw data from protein-binding experimentsdescribed in Example 3. FIG. 18A depicts the array from “plate 0” probedwith FKBP-Cy5. FIGS. 18B and 18C depict the array from “Plate 1” probedwith FKBP-GST (red channel only). FIGS. 18D and 18E depict the arrayfrom “Plate 1” probed with FKBP-GST (red and green channels).

FIG. 19 depicts exemplary raw data from BrdU Cytoblot (cell-based assay)experiments described in Example 3. FIGS. 19A and 19B depict replicateassays of “Plate 0” by the BrdU cytoblot assay. FIGS. 19C and 19D depictreplicate assays of “Plate 1” by the BrdU cytoblot assay. FIGS. 19E and19F depict multiplicative overlays of the replicate “Plate 0” and “Plate1” assays depicted in FIGS. 19A-B and FIGS. 19C-D, respectively.

FIG. 20 depicts results of an exemplary genistein suppressor assay, asdescribed in Example 3. FIGS. 20A and 20B represent duplicate assays of“Plate 1”.

FIG. 21A depicts a 384-well-plate from a BrdU incorporation inhibitionassay, identifying wells containing compounds inhibiting BrdUincorporation in cells.

FIG. 21B depicts a 384-well-plate from a Genistein suppression assay,identifying wells containing compounds than can suppress the ability ofgenistein to inhibit BrdU incorporation.

FIG. 22 depicts an example of LC and MS results from “hits” generatedfrom cytoblot assay of exemplary inventive compounds.

FIG. 23 depicts a robotic 384 pin arrayer.

FIG. 24 depicts small molecule printing.

FIG. 25 depicts a small molecule microarraying robot.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

As discussed above, there remains a need for the development of noveltherapeutic agents and agents capable of elucidating biologicalfunctions. In one aspect, the present invention provides novel compoundsof general formula (I), and methods for the synthesis thereof, whichcompounds are useful, for example, as DNA synthase inhibitors and Eg5inhibitors, and thus are useful for the treatment of, for example,proliferative diseases and cancer. In certain embodiments, the inventivecompounds are additionally useful as tools to probe biological function.

General Description of Compounds of the Invention

As detailed above, in one aspect of the invention, noveldihydropyrancarboxamides having the following structure (I) areprovided:

wherein R¹-R⁴ are each independently hydrogen or an aliphatic,heteroaliphatic, aryl, heteroaryl, aliphatic(aryl),heteroaliphatic(aryl), aliphatic(heteroaryl) orheteroaliphatic(heteroaryl) moiety;

R⁵ and R⁶ are each independently hydrogen or an aliphatic,heteroaliphatic, aryl, heteroaryl, alkylaryl, or alkylheteroaryl moiety,and wherein R⁵ and R⁶, taken together, may form a cyclic aliphatic,heteroaliphatic, aliphatic(aryl), heteroaliphatic(aryl),aliphatic(heteroaryl) or heteroaliphatic(heteroaryl) moiety, or an arylor heteroaryl moiety;

wherein each of the foregoing aliphatic and heteroaliphatic moieties maybe substituted or unsubstituted, cyclic or acyclic, saturated orunsaturated or linear or branched; and each of the foregoing aryl,heteroaryl, aliphatic(aryl), heteroaliphatic(aryl),aliphatic(heteroaryl) or heteroaliphatic(heteroaryl) moieties may besubstituted or unsubstituted; and

pharmaceutically acceptable derivatives thereof.

In one exemplary subset of the invention, compounds having the followingstructure (II) are provided:

wherein R¹-R⁴ are each independently hydrogen or an aliphatic,heteroaliphatic, aryl, heteroaryl, aliphatic(aryl),heteroaliphatic(aryl), aliphatic(heteroaryl) orheteroaliphatic(heteroaryl) moiety;

R⁵ and R⁶ are each independently hydrogen or an aliphatic,heteroaliphatic, aryl, heteroaryl, alkylaryl, or alkylheteroaryl moiety,and wherein R⁵ and R⁶, taken together, may form a cyclic aliphatic,heteroaliphatic, aliphatic(aryl), heteroaliphatic(aryl),aliphatic(heteroaryl) or heteroaliphatic(heteroaryl) moiety, or an arylor heteroaryl moiety;

wherein each of the foregoing aliphatic and heteroaliphatic moieties maybe substituted or unsubstituted, cyclic or acyclic, saturated orunsaturated or linear or branched; and each of the foregoing aryl,heteroaryl, aliphatic(aryl), heteroaliphatic(aryl),aliphatic(heteroaryl) or heteroaliphatic(heteroaryl) moieties may besubstituted or unsubstituted; and

pharmaceutically acceptable derivatives thereof.

In still other subsets of the invention, compounds are provided in whichthe conjugated carboxylate (R⁴) is functionalized. In still othersubsets of the invention, compounds are provided in which the carbonylis functionalized. In still other subsets of the invention, compoundsare provided in which R¹ is a solid support linked through a silyllinker as described in Examples 1 and 2 herein. In still other subsetsof the invention, compounds are provided in which R² is a solid supportlinked through a silyl linker as described in Examples 1 and 2 herein.In further subsets of the invention, compounds having functionalizationat two or more of these sites are provided. In still other subsets ofthe invention, compounds having functionalization at each of these sitesare provided. In certain other subsets of the invention, compounds areprovided as described using the reagents detailed in Example 1.

In another embodiment of the invention, the inventive compounds areprovided as a collection and thus may be provided as a collection of twoor more of any of the compounds as detailed above or as describedherein. In certain embodiments, the collection is provided in arrayformat. In certain other embodiments, the collection is provided inarray format on a glass slide. In still other embodiments, thecollection comprises at least 100 compounds. In yet other embodiments,the collection comprises at least 1,000, 2,000 or 10,000 compounds.

2) Featured Classes of Compounds

In certain embodiments, the present invention defines certain classes ofcompounds which are of special interest.

For example, one class of compounds of special interest includes thosecompounds of the invention as described above and in certain subclassesherein, in which R¹ is hydrogen, Z or an alkyl, heteroalkyl, aryl orheteroaryl moiety substituted with Z, wherein Z is hydrogen,—(CH₂)_(q)OR^(Z), —(CH₂)_(q)SR^(Z), —(CH₂)_(q)N(Re)₂, —(C═O)R^(Z),—(C═O)N(R^(Z))₂, or an aliphatic, heteroaliphatic, aryl, heteroaryl,-(aliphatic)aryl, -(aliphatic)heteroaryl, -(heteroaliphatic)aryl, or-(heteroaliphatic)heteroaryl moiety, wherein q is 0-4, and wherein eachoccurrence of R^(Z) is independently hydrogen, a protecting group, asolid support unit, or an aliphatic, heteroaliphatic, aryl, heteroaryl,-(aliphatic)aryl, -(aliphatic)heteroaryl, -(heteroaliphatic)aryl, or-(heteroaliphatic)heteroaryl moiety; wherein each of the foregoing alkylor heteroalkyl moieties may be substituted or unsubstituted, linear orbranched, cyclic or acyclic, saturated or unsaturated; and wherein eachof the foregoing aryl, heteroaryl, -(alkyl)aryl, -(alkyl)heteroaryl,(heteroalkyl)aryl, or -heteroalkyl)heteroaryl moieties may besubstituted or unsubstituted.

Another class of compounds of special interest includes those compoundsof the invention as described above and in certain subclasses herein, inwhich R² is hydrogen, Z or an alkyl, heteroalkyl, aryl or heteroarylmoiety substituted with Z, wherein Z is hydrogen, —(CH₂)_(q)OR^(Z),—(CH₂)_(q)SR^(Z), —(CH₂)_(q)N(R^(Z))₂, —(C═O)R^(Z), —(C═O)N(R^(Z))₂, oran aliphatic, heteroaliphatic, aryl, heteroaryl, -(aliphatic)aryl,-(aliphatic)heteroaryl, -(heteroaliphatic)aryl, or-heteroaliphatic)heteroaryl moiety, wherein q is 0-4, and wherein eachoccurrence of R^(Z) is independently hydrogen, a protecting group, asolid support unit, or an aliphatic, heteroaliphatic, aryl, heteroaryl,-(aliphatic)aryl, -(aliphatic)heteroaryl, -(heteroaliphatic)aryl, or-(heteroaliphatic)heteroaryl moiety; wherein each of the foregoing alkylor heteroalkyl moieties may be substituted or unsubstituted, linear orbranched, cyclic or acyclic, saturated or unsaturated; and wherein eachof the foregoing aryl, heteroaryl, -(alkyl)aryl, -(alkyl)heteroaryl,-(heteroalkyl)aryl, or -(heteroalkyl)heteroaryl moieties may besubstituted or unsubstituted.

Another class of compounds of special interest includes those compoundsof the invention as described above and in certain subclasses herein, inwhich R³ is an alkyl, heteroalkyl, aryl, heteroaryl, -(alkyl)aryl,-(alkyl)heteroaryl, -(heteroalkyl)aryl, or -(heteroalkyl)heteroarylmoiety; wherein each of the foregoing alkyl or heteroalkyl moieties maybe substituted or unsubstituted, linear or branched, cyclic or acyclic,saturated or unsaturated; and wherein each of the foregoing aryl,heteroaryl, -(alkyl)aryl, -(alkyl)heteroaryl, -(heteroalkyl)aryl, or-(heteroalkyl)heteroaryl moieties may be substituted or unsubstituted.

Another class of compounds of special interest includes those compoundsof the invention as described above and in certain subclasses herein, inwhich R⁴ is hydrogen or an alkyl, heteroalkyl, aryl, heteroaryl,-(alkyl)aryl, -(alkyl)heteroaryl, -(heteroalkyl)aryl, or-(heteroalkyl)heteroaryl moiety; wherein each of the foregoing alkyl orheteroalkyl moieties may be substituted or unsubstituted, linear orbranched, cyclic or acyclic, saturated or unsaturated; and wherein eachof the foregoing aryl, heteroaryl, -(alkyl)aryl, -(alkyl)heteroaryl,-(heteroalkyl)aryl, or -(heteroalkyl)heteroaryl moieties may besubstituted or unsubstituted.

Another class of compounds of special interest includes those compoundsof the invention as described above and in certain subclasses herein, inwhich R⁵ and R⁶ are each independently hydrogen or an alkyl,heteroalkyl, aryl, heteroaryl, -(alkyl)aryl, -(alkyl)heteroaryl,-(heteroalkyl)aryl, or -(heteroalkyl)heteroaryl moiety; or wherein R⁵and R⁶, taken together, form a substituted or unsubstituted, saturatedor unsaturated cyclic moiety comprising 5-12 carbon atoms, 0-5 oxygenatoms, 0-5 sulfur atoms and 1-5 nitrogen atoms; and wherein each of theforegoing alkyl or heteroalkyl moieties may be substituted orunsubstituted, linear or branched, cyclic or acyclic, saturated orunsaturated; and wherein each of the foregoing aryl, heteroaryl,-(alkyl)aryl, -(alkyl)heteroaryl, -(heteroalkyl)aryl, or-(heteroalkyl)heteroaryl moieties may be substituted or unsubstituted.

The following compounds are illustrative of certain of the compoundsdescribed generally and in classes and subclasses herein:

A number of important subclasses of each of the foregoing classesdeserve separate mention; these subclasses include subclasses of each ofthe foregoing classes in which:

i) compounds of the invention as described above and herein wherein R¹is hydrogen, lower alkyl, a substituted or unsubstituted phenyl orslower alkyl)phenyl moiety, —(CH₂)_(n)OR^(z), -[(CH₂)_(n)O]_(m)R^(z), or—(CH₂)_(n)—Ar—(CH₂)_(m)OR^(z); wherein n and m are each independentlyintegers from 1-6, Ar represents a substituted or unsubstituted aryl orheteroaryl moiety, and R^(z) is independently hydrogen, a protectinggroup, a solid support unit, or an aliphatic, heteroaliphatic, aryl,heteroaryl, -(aliphatic)aryl, -(aliphatic)heteroaryl,-(heteroaliphatic)aryl, or -(heteroaliphatic)heteroaryl moiety; whereineach of the foregoing alkyl or heteroalkyl moieties may be substitutedor unsubstituted, linear or branched, cyclic or acyclic, saturated orunsaturated; and wherein each of the foregoing aryl, heteroaryl,-(alkyl)aryl, -(alkyl)heteroaryl, -(heteroalkyl)aryl, or-(heteroalkyl)heteroaryl moieties may be substituted or unsubstituted;

ii) compounds of the invention as described above and herein wherein R¹is attached to a solid support;

iii) compounds of the invention as described above and herein wherein R¹is —(CH₂)_(n)OR^(z), [(CH₂)_(n)O]_(m)R^(z), or—(CH₂)_(n)—Ar—(CH₂)_(m)OR^(z); wherein n and m are each independentlyintegers from 1-6, Ar represents a substituted or unsubstituted aryl orheteroaryl moiety, and R^(z) is hydrogen, a protecting group or a solidsupport unit;

iv) compounds of the invention as described above and herein wherein R¹is —(CH₂)_(n)OR^(z), —[(CH₂)_(n)O]_(m)R^(z), or—(CH₂)_(n)—Ar—(CH₂)_(m)OR^(z); wherein n and m are each independentlyintegers from 1-6, Ar represents a substituted or unsubstituted aryl orheteroaryl moiety, and R^(z) is a solid support unit linked to O througha silyl linker; or heteroalkyl, or substituted or unsubstituted aryl,heteroaryl, -(alkyl)aryl, -(alkyl)heteroaryl, -(heteroalkyl)aryl, or-(heteroalkyl)heteroaryl;

xviii) compounds as described above and herein wherein R³ has one of thestructures:

xix) compounds as described above and herein wherein R³ has one of thestructures:

xx) compounds as described above and herein wherein R⁴ is hydrogen,alkyl, or heteroalkyl;

xxi) compounds as described above and herein wherein R⁴ is hydrogen; and

xxii) compounds as described above and herein wherein —NR⁵R⁶ has one ofthe structures:

v) compounds of the invention as described above and herein wherein R¹is hydrogen, or lower alkyl;

vi) compounds of the invention as described above and herein wherein R¹is ethyl;

vii) compounds of the invention as described above and herein wherein R¹is hydrogen, ethyl, or has one of the structures:

wherein R^(z) is hydrogen, a protecting group or a solid support unit;

viii) compounds of the invention as described above and herein whereinR¹ is hydrogen, ethyl, or has one of the structures:

wherein R^(z) is hydrogen, a protecting group or a solid support unit;

ix) compounds of the invention as described above and herein wherein R²is hydrogen, lower alkyl, a substituted or unsubstituted phenyl or-(lower alkyl)phenyl moiety, —(CH₂)_(n)OR^(z), —[(CH₂)_(n)O]_(m)R^(z),—(CH₂)_(n)—Ar—(CH₂)_(m)OR^(z); wherein n and m are each independentlyintegers from 1-6, Ar represents a substituted or unsubstituted aryl orheteroaryl moiety, and R^(z) is independently hydrogen, a protectinggroup, a solid support unit, or an aliphatic, heteroaliphatic, aryl,heteroaryl, -(aliphatic)aryl, -(aliphatic)heteroaryl,-(heteroaliphatic)aryl, or -(heteroaliphatic)heteroaryl moiety; whereineach of the foregoing alkyl or heteroalkyl moieties may be substitutedor unsubstituted, linear or branched, cyclic or acyclic, saturated orunsaturated; and wherein each of the foregoing aryl, heteroaryl,-(alkyl)aryl, -(alkyl)heteroaryl, -(heteroalkyl)aryl, or-(heteroalkyl)heteroaryl moieties may be substituted or unsubstituted;

x) compounds of the invention as described above and herein wherein R²is attached to a solid support;

xi) compounds of the invention as described above and herein wherein R²is —(CH₂)_(n)OR^(z), —[(CH₂)_(n)O]_(m)R^(z), or—(CH₂)_(n)—Ar—(CH₂)_(m)OR^(z); wherein n and m are each independentlyintegers from 1-6, Ar represents a substituted or unsubstituted aryl orheteroaryl moiety, and R^(z) is hydrogen, a protecting group or a solidsupport unit;

xii) compounds of the invention as described above and herein wherein R²is —(CH₂)_(n)OR^(z), —[(CH₂)_(n)O]_(m)R^(z), or—(CH₂)_(n)—Ar—(CH₂)_(m)OR^(z); wherein n and m are each independentlyintegers from 1-6, Ar represents a substituted or unsubstituted aryl orheteroaryl moiety, and R^(z) is a solid support unit linked to O througha silyl linker;

xiii) compounds of the invention as described above and herein whereinR² is hydrogen or lower alkyl;

xiv) compounds of the invention as described above and herein wherein R²is hydrogen, methyl or ethyl;

xv) compounds of the invention as described above and herein wherein R²is ethyl;

xvi) compounds as described above and herein wherein R² is hydrogen orhas one of the structures:

wherein R^(z) is hydrogen, a protecting group or a solid support unit;

xvii) compounds of the invention as described above and herein whereinR³ is substituted or unsubstituted, cyclic or acyclic, linear orbranched, saturated or unsaturated alkyl

As the reader will appreciate, compounds of particular interest include,among others, those which share the attributes of one or more of theforegoing subclasses. Some of those subclasses are illustrated by thefollowing sorts of compounds:

I) Compounds of the Formula:

wherein R², R³, R⁴, R⁵, R⁶ and R^(Z) are as described in classes andsubclasses herein; and Y is a substituted or unsubstituted, cyclic oracyclic, linear or branched, saturated or unsaturated aliphatic orheteroaliphatic moiety, or a substituted or unsubstituted aryl,heteroaryl, -(aliphatic)aryl, -(aliphatic)heteroaryl,-(heteroaliphatic)aryl, or -(heteroaliphatic)heteroaryl moiety; whereineach of the foregoing alkyl or heteroalkyl moieties may be substitutedor unsubstituted, linear or branched, cyclic or acyclic, saturated orunsaturated; and wherein each of the foregoing aryl, heteroaryl,(aliphatic)aryl, -(aliphatic)heteroaryl, -(heteroaliphatic)aryl, or-(heteroaliphatic)heteroaryl moieties may be substituted orunsubstituted.

In certain exemplary embodiments, Y is an alkyl, heteroalkyl, aryl,heteroaryl, -(alkyl)aryl, -(alkyl)heteroaryl, -(heteroalkyl)aryl, or-(heteroalkyl)heteroaryl moiety; R^(Z) is hydrogen, a protecting groupor a solid support unit; R², R³ and R⁴ are each independently hydrogenor an alkyl, heteroalkyl, aryl, heteroaryl, -(alkyl)aryl,-(alkyl)heteroaryl, -(heteroalkyl)aryl, or -(heteroalkyl)heteroarylmoiety; and R⁵ and R⁶ are each independently hydrogen or an alkyl,heteroalkyl, aryl, heteroaryl, -(alkyl)aryl, -(alkyl)heteroaryl,-(heteroalkyl)aryl, or -(heteroalkyl)heteroaryl moiety; or wherein R⁵and R⁶, taken together, form a substituted or unsubstituted, saturatedor unsaturated cyclic moiety comprising 5-12 carbon atoms, 0-5 oxygenatoms, 0-5 sulfur atoms and 1-5 nitrogen atoms; and wherein each of theforegoing alkyl or heteroalkyl moieties may be substituted orunsubstituted, linear or branched, cyclic or acyclic, saturated orunsaturated; and wherein each of the foregoing aryl, heteroaryl,-(alkyl)aryl, -(alkyl)heteroaryl, -(heteroalkyl)aryl, or(heteroalkyl)heteroaryl moieties may be substituted or unsubstituted.

In certain embodiments, R^(z)—Y— together represents a moiety having thestructure:

wherein R^(z) is hydrogen, a protecting group or a solid support unit.

In certain exemplary embodiments, R^(z)—Y— together represents a moietyhaving the structure:

wherein R^(z) is hydrogen, a protecting group or a solid support unit.

II) Compounds of the Formula:

wherein R¹, R³, R⁴, R⁵, R⁶ and R^(Z) are as described in classes andsubclasses herein; and X is a substituted or unsubstituted, cyclic oracyclic, linear or branched, saturated or unsaturated aliphatic orheteroaliphatic moiety, or a substituted or unsubstituted aryl,heteroaryl, -(aliphatic)aryl, -(aliphatic)heteroaryl,-(heteroaliphatic)aryl, or -(heteroaliphatic)heteroaryl moiety; whereineach of the foregoing alkyl or heteroalkyl moieties may be substitutedor unsubstituted, linear or branched, cyclic or acyclic, saturated orunsaturated; and wherein each of the foregoing aryl, heteroaryl,(aliphatic)aryl, -(aliphatic)heteroaryl, -(heteroaliphatic)aryl, or-(heteroaliphatic)heteroaryl moieties may be substituted orunsubstituted.

In certain exemplary embodiments, X is an alkyl, heteroalkyl, aryl,heteroaryl, -(alkyl)aryl, -(alkyl)heteroaryl, -(heteroalkyl)aryl, or-(heteroalkyl)heteroaryl moiety; R^(Z) is hydrogen, a protecting groupor a solid support unit; R², R³ and R⁴ are each independently hydrogenor an alkyl, heteroalkyl, aryl, heteroaryl, -(alkyl)aryl,-(alkyl)heteroaryl, -(heteroalkyl)aryl, or -(heteroalkyl)heteroarylmoiety; and R⁵ and R⁶ are each independently hydrogen or an alkyl,heteroalkyl, aryl, heteroaryl, -(alkyl)aryl, -(alkyl)heteroaryl,-(heteroalkyl)aryl, or -(heteroalkyl)heteroaryl moiety; or wherein R⁵and R⁶, taken together, form a substituted or unsubstituted, saturatedor unsaturated cyclic moiety comprising 5-12 carbon atoms, 0-5 oxygenatoms, 0-5 sulfur atoms and 1-5 nitrogen atoms; and wherein each of theforegoing alkyl or heteroalkyl moieties may be substituted orunsubstituted, linear or branched, cyclic or acyclic, saturated orunsaturated; and wherein each of the foregoing aryl, heteroaryl,-(alkyl)aryl, -(alkyl)heteroaryl, -(heteroalkyl)aryl, or-(heteroalkyl)heteroaryl moieties may be substituted or unsubstituted.

In certain embodiments, R^(z)—X— together represents a moiety having thestructure:

wherein R^(z) is hydrogen, a protecting group or a solid support unit.

III) Compounds of the Formula:

wherein R¹, R², R⁴, R⁵ and R⁶ are as described in classes and subclassesherein; and Ar is a substituted or unsubstituted aryl or heteroarylmoiety.

In certain exemplary embodiments, R¹ and R² are each independentlyhydrogen, Z or an alkyl, heteroalkyl, aryl or heteroaryl moietysubstituted with Z, wherein Z is hydrogen, —(CH₂)_(q)OR^(Z),—(CH₂)_(q)SR^(Z), —(CH₂)_(q)N(R^(Z))₂, —(C═O)R^(Z), —(C═O)N(R^(Z))₂, oran aliphatic, heteroaliphatic, aryl, heteroaryl, -(aliphatic)aryl,-(aliphatic)heteroaryl, -(heteroaliphatic)aryl, or-heteroaliphatic)heteroaryl moiety, wherein q is 0-4, and wherein eachoccurrence of R^(Z) is independently hydrogen, a protecting group, asolid support unit, or an aliphatic, heteroaliphatic, aryl, heteroaryl,-(aliphatic)aryl, -(aliphatic)heteroaryl, -(heteroaliphatic)aryl, or-(heteroaliphatic)heteroaryl moiety; R⁴ is hydrogen or an alkyl,heteroalkyl, aryl, heteroaryl, -(alkyl)aryl, -(alkyl)heteroaryl,-(heteroalkyl)aryl, or -(heteroalkyl)heteroaryl moiety; and R⁵ and R⁶are each independently hydrogen or an alkyl, heteroalkyl, aryl,heteroaryl, -(alkyl)aryl, -(alkyl)heteroaryl, -(heteroalkyl)aryl, or-(heteroalkyl)heteroaryl moiety; or wherein R⁵ and R⁶, taken together,form a substituted or unsubstituted, saturated or unsaturated cyclicmoiety comprising 5-12 carbon atoms, 0-5 oxygen atoms, 0-5 sulfur atomsand 1-5 nitrogen atoms; and wherein each of the foregoing aliphatic,heteroaliphatic, alkyl or heteroalkyl moieties may be substituted orunsubstituted, linear or branched, cyclic or acyclic, saturated orunsaturated; and wherein each of the foregoing aryl, heteroaryl,-(alkyl)aryl, -(alkyl)heteroaryl, -(heteroalkyl)aryl,-heteroalkyl)heteroaryl, -(aliphatic)aryl, -(aliphatic)heteroaryl,-(heteroaliphatic)aryl, or -(heteroaliphatic)heteroaryl moieties may besubstituted or unsubstituted.

In certain exemplary embodiments, Ar is a moiety having the structure:

which may further be substituted with one or more occurrences of anysubstitutents described in the Definitions above.

In certain other exemplary embodiments, Ar is a moiety having thestructure:

which may further be substituted with one or more occurrences of anysubstitutents described in the Definitions above.

IV) Compounds of the Formula:

wherein R¹, R², R³, R⁵ and R⁶ are as described in classes and subclassesherein.

In certain exemplary embodiments, R¹ and R² are each independentlyhydrogen, Z or an alkyl, heteroalkyl, aryl or heteroaryl moietysubstituted with Z, wherein Z is hydrogen, —(CH₂)_(q)OR^(Z),—(CH₂)_(q)SR^(Z), —(CH₂)_(q)N(R^(Z))₂, —(C═O)R^(Z), —(C═O)N(R^(Z))₂, oran aliphatic, heteroaliphatic, aryl, heteroaryl, -(aliphatic)aryl,-(aliphatic)heteroaryl, -(heteroaliphatic)aryl, or-(heteroaliphatic)heteroaryl moiety, wherein q is 0-4, and wherein eachoccurrence of R^(Z) is independently hydrogen, a protecting group, asolid support unit, or an aliphatic, heteroaliphatic, aryl, heteroaryl,-(aliphatic)aryl, -(aliphatic)heteroaryl, -(heteroaliphatic)aryl, or-(heteroaliphatic)heteroaryl moiety; R³ is hydrogen or an alkyl,heteroalkyl, aryl, heteroaryl, -(alkyl)aryl, -(alkyl)heteroaryl,-(heteroalkyl)aryl, or -(heteroalkyl)heteroaryl moiety; R⁵ and R⁶ areeach independently hydrogen or an alkyl, heteroalkyl, aryl, heteroaryl,-(alkyl)aryl, -(alkyl)heteroaryl, -(heteroalkyl)aryl, or-(heteroalkyl)heteroaryl moiety; or wherein R⁵ and R⁶, taken together,form a substituted or unsubstituted, saturated or unsaturated cyclicmoiety comprising 5-12 carbon atoms, 0-5 oxygen atoms, 0-5 sulfur atomsand 1-5 nitrogen atoms; and wherein each of the foregoing aliphatic,heteroaliphatic, alkyl or heteroalkyl moieties may be substituted orunsubstituted, linear or branched, cyclic or acyclic, saturated orunsaturated; and wherein each of the foregoing aryl, heteroaryl,-(alkyl)aryl, -(alkyl)heteroaryl, -(heteroalkyl)aryl,-(heteroalkyl)heteroaryl, -(aliphatic)aryl, -(aliphatic)heteroaryl,-(heteroaliphatic)aryl, or -(heteroaliphatic)heteroaryl moieties may besubstituted or unsubstituted.

It will be appreciated that some of the foregoing classes and subclassesof compounds can exist in various isomeric forms. The inventionencompasses the compounds as individual isomers substantially free ofother isomers and alternatively, as mixtures of various isomers, e.g.,racemic mixtures of stereoisomers. Additionally, when R¹, R², R³, R⁴, R⁵and/or R⁶ comprise a double bond, the invention encompasses both (Z) and(E) double bond isomers unless otherwise specifically designated. Theinvention also encompasses tautomers of specific compounds as describedabove. In addition to the above-mentioned compounds per se, thisinvention also encompasses pharmaceutically acceptable derivatives ofthese compounds and compositions comprising one or more compounds of theinvention and one or more pharmaceutically acceptable excipients oradditives.

Synthetic Methodology

In yet another aspect of the present invention, novel methods for thesynthesis of the novel dihydropyrancarboxamides as described herein areprovided.

According to the present invention, any available techniques can be usedto make or prepare the inventive dihydropyrancarboxamides orcompositions including them. For example, combinatorial techniques,parallel synthesis and/or solid phase synthetic methods such as thosediscussed in detail below may be used. Alternatively, the inventivecompounds may be prepared using any of a variety of solution phasesynthetic methods known in the art (e.g., one compounds at a time).

In certain exemplary embodiments, the method takes advantage ofefficient catalytic asymmetric heterocycloaddition reactions as depictedin FIG. 2 (see, a) D. A. Evans, J. S. Johnson, E. J. Olhava, J. Am.Chem. Soc. 2000, 122, 1635-1649; b) D. A. Evans, E. J. Olhava, J. S.Johnson, J. M. Janey, Angew. Chem. 1998, 110, 3554-3557; Angew. Chem.Int. Ed. 1998, 37, 3372-3375; c) J. Thorhauge, M. Johannsen, K. A.Jorgensen, Angew. Chem. 1998, 110, 2543-2546; Angew. Chem. Int. Ed.1998, 37, 2404-2406; d) H. E. Balckwell, L. Pérez, R. A. Stavenger, J.A. Tallarico, E. Cope Eatough, M. A. Foley, S. L. Schreiber, Chem. Biol.2001, 1167-1182; e) P. A. Clemmons, A. N. Koehler, B. K. Wagner, T. G.,Sprigings, D. R. Spring, R. W. King, S. L. Schreiber, M. A. Foley, Chem.Biol. 2001, 1183-1195; and f) R. A. Stavenger, S. L. Schreiber, Angew.Chem. Int. Ed. 2001, 40(18), 3417-3421). In certain embodiments,following the heterocycloaddition reaction, as depicted in FIG. 2, avariety of diversity generating reactions may be performed to completethe synthesis of each member of the library of compounds.

In certain exemplary embodiments, according to the method of the presentinvention, a core structure can be provided, wherein the core structureis synthesized by the method comprising:

providing a vinyl ether having the structure:

providing an unsaturated ketoester having the structure:

subjecting the vinyl ether and the unsaturated ketoester to suitableconditions to generate a scaffold having the core structure:

wherein R¹ and R² are each independently hydrogen or an aliphatic,heteroaliphatic, aryl, heteroaryl, alkylaryl or alkylheteroaryl moiety;wherein one of R¹ or R² is attached to a solid support;

R³ and R⁴ are each independently hydrogen or an aliphatic,heteroaliphatic, aryl, heteroaryl, alkylaryl or alkylheteroaryl moiety;

R_(A) is hydrogen or is an aliphatic, heteroaliphatic, aryl, heteroaryl,alkylaryl, or alkylheteroaryl moiety;

wherein each of the foregoing aliphatic and heteroaliphatic moieties maybe substituted or unsubstituted, cyclic or acyclic, saturated orunsaturated or linear or branched; and each of the foregoing aryl,heteroaryl, alkylaryl or alkylheteroaryl moieties may be substituted orunsubstituted.

In certain embodiments, R¹ or R² is attached to a solid support via asilyl linker.

It will be appreciated that the synthetic methods, as described herein,may utilize a variety of protecting groups (e.g. O, S, or N protectinggroups) to temporarily block a particular functional group so that areaction can be carried out selectively at another reactive site in amultifunctional compound. One of ordinary skill in the art willrecognize that, in addition to the specific protecting groups describedin the Examples herein, a variety of well-known protecting groups in theart of organic synthesis can also be utilized as detailed in Greene andWuts, Protective Groups in Organic Synthesis, Third Edition, John Wiley& Sons, New York: 1999, the entire contents of which are herebyincorporated by reference.

Once the core structure is prepared, as detailed above, one or morecompounds can be synthesized via combinatorial techniques, or bysynthesizing one compound at a time, by diversifying at particularfunctional groups. Thus, in another embodiment, the method furthercomprises functionalizing the core structure (III) at one or more sitesto generate compounds having the structures (Ia):

wherein R¹-R⁶ are as defined in classes and subclasses herein; and oneof R¹ or R² is attached to a solid support.

In certain embodiments, the method further comprises functionalizing thecore structure (III) at one or more sites to generate compounds havingthe structures (IIa):

and/or enantiomer thereof;

wherein R¹-R⁶ are as defined in classes and subclasses herein; and oneof R¹ or R² is attached to a solid support.

In certain embodiments, the carboxylic ester moiety can be converted,among others, to an acid halide, amide, anhydride, diketone, imide ornitrile moiety; the conjugated carboxyl moiety can be functionalized viaconjugate addition and can be diversified using oxygen, sulfur, nitrogenor carbon nucleophiles, to name a few. In addition, where thesubstitutents R_(A), R¹, R², R³, R⁴, R⁵ and/or R⁶ comprise an aryl orheteroaryl group, such aryl or heteroaryl group may be furtherdiversified by introducing additional functionalities according tomethods known in the art.

In certain embodiments, the method further comprises cleaving the corestructure (III) from the solid support to which it is attached eitherbefore or after chemical derivatization.

In certain exemplary embodiments, the carboxylic ester CO₂R^(A) can bederivatized to form the corresponding amido compound having thestructure (Ia):

wherein R¹ and R² are each independently hydrogen or an aliphatic,heteroaliphatic, aryl, heteroaryl, alkylaryl or alkylheteroaryl moiety;wherein one of R¹ or R² is attached to a solid support;

R³ and R⁴ are each independently hydrogen or an aliphatic,heteroaliphatic, aryl, heteroaryl, alkylaryl or alkylheteroaryl moiety;

R⁵ and R⁶ are each independently hydrogen or an aliphatic,heteroaliphatic, aryl, heteroaryl, alkylaryl, or alkylheteroaryl moiety,and wherein R⁵ and R⁶, taken together, may form a cyclic aliphatic,heteroaliphatic, aliphatic(aryl), heteroaliphatic(aryl),aliphatic(heteroaryl) or heteroaliphatic(heteroaryl) moiety, or an arylor heteroaryl moiety;

wherein each of the foregoing aliphatic and heteroaliphatic moieties maybe substituted or unsubstituted, cyclic or acyclic, saturated orunsaturated or linear or branched; and each of the foregoing aryl,heteroaryl, alkylaryl or alkylheteroaryl moieties may be substituted orunsubstituted.

In certain exemplary embodiments, synthetic transformation of thecarboxylic moiety —CO₂R_(A) is achieved by conversion of the estermoiety to the corresponding carboxylic acid, followed reaction with asuitable amine under conditions suitable to effect amide formation.Examples of amines suitable for practicing the invention include, butare not limited to

In certain exemplary embodiments, conjugated carboxylatefunctionalization is achieved using furfuryl mercaptan,3-(trifluoromethyl)benzyl mercaptan, 3-methyl-1-buranethiol,4-methoxy-alpha-toluenethiol, benzyl mercaptan, 2-(tertbutyldimethylsiloxy)ethylmercaptan, cyclopentanethiol, or a skip codol.In certain embodiments, amine (nitrogen functionalization) is achievedusing benzoyl chloride, benzyl isocyanate, ethyl isocyanate,thiophene-2-carbonyl chloride, 3-(methylthio)propionaldehyde, undecanal,cyclopropanecarboxaldehyde, or a skip codon. In certain embodiments,ketone functionalization is achieved using p-toluenesulfonhydrazide,dansyl hydrazine, methoxyamine hydrochloride, o-Benzylhydroxylaminehydrochloride, Carboxymethoxylamine hemihydrochloride,p-Methoxybenzensulfonylhydrazide, 4-Nitrophenylhydrazine or a skipcodon.

Although certain exemplary diversification reactions and reagents aredescribed in more detail herein, it will be appreciated that the presentinvention is intended to encompass equivalent diversification reactionswithin the arsenal of synthetic organic chemistry that can be utilizedto diversify the inventive scaffold as described herein (See, generally,March, Advanced Organic Chemistry, John Wiley & Sons, 1992; and“Comprehensive Organic Transformations, a guide to functional grouppreparations”, Richard C. Larock, VCH publishers, 1999; the entirecontents of which are incorporated herein by reference). For example,although certain reagents for amide formation are described in theexamples (e.g., 2-methoxy-ethylamine), it will be appreciated that otherderivatives can be utilized (e.g., 2-ethoxy-ethylamine,2-propoxy-ethylamine, etc.), including, but not limited to, homologuesand other similarly substituted moieties. These additional examples arenot intended to limit the scope of the invention; rather they areprovided to exemplify the broad utility of the inventive scaffold in theemployment of a variety of diversification reactions and reagents.

In certain embodiments, the method further comprises cleaving thestructure (Ia) from the solid support to which it is attached to give acompound having the structure (I):

wherein R¹-R⁴ are each independently hydrogen or an aliphatic,heteroaliphatic, aryl, heteroaryl, alkylaryl or alkylheteroaryl moiety;

R⁵ and R⁶ are each independently hydrogen or an aliphatic,heteroaliphatic, aryl, heteroaryl, alkylaryl, or alkylheteroaryl moiety,and wherein R⁵ and R⁶, taken together, may form a cyclic aliphatic,heteroaliphatic, aliphatic(aryl), heteroaliphatic(aryl),aliphatic(heteroaryl) or heteroaliphatic(heteroaryl) moiety, or an arylor heteroaryl moiety;

wherein each of the foregoing aliphatic and heteroaliphatic moieties maybe substituted or unsubstituted, cyclic or acyclic, saturated orunsaturated or linear or branched; and each of the foregoing aryl,heteroaryl, alkylaryl or alkylheteroaryl moieties may be substituted orunsubstituted.

In one exemplary embodiment, as depicted in FIG. 1, and as described inExample 1, a library was synthesized on 500-600 μm high capacitypolystyrene beads functionalized with a trialkylsilyl linker (See also,Scheme 1) (P. A. Clemmons, A. N. Koehler, B. K. Wagner, T. G.,Sprigings, D. R. Spring, R. W. King, S. L. Schreiber, M. A. Foley, Chem.Biol. 2001, 1183-1195; see also Example 2, herein).

For example, as depicted in Scheme 1, disoprolyalkylsilyl-functionalizedresin 1a can be activated by treatment with excess triflic acid to form1b. Alcohol compound ROH can then be trapped onto the activated resin inthe presence of excess 2,6-lutidine to generate the corresponding silylether 1c. After subjecting —R to suitable reaction conditions to effectthe desired synthetic transformations, the substrate may be cleaved fromthe solid support 1d by reaction with HF, followed by quenching ofexcess HF with methoxytrimethylsilane (TMSOMe), to give the desiredlibrary members. In certain embodiments, for the library synthesis,building blocks can be selected that reacted in good yield and, as agroup, possess diverse physical characteristics. Exemplary buildingblocks suitable for practicing the invention are depicted in FIG. 3. Oneof ordinary skill in the art will appreciate that other building blocksmay be used.

In but one exemplary embodiment, the library was prepared as atriplicate copy (3 bead per library member), arrayed in 384-well plates,and detached from the solid-support with HF-pyridine. Evaporation of thecleavage cocktail and resuspension in DMF or DMSO afforded 4320 stocksolutions for biological screening.

Research Uses

According to the present invention, the inventive compounds may beassayed in any of the available assays known in the art for identifyingcompounds having a biological activity of interest. For example, theassay may be cellular or non-cellular, in vivo or in vitro, etc. Anyassay format may be used to screen the inventive compounds (e.g.,formats amenable to high-throughput screening). Examples of biologicalactivity include, but are not limited to, binding activity or biologicalactivity against target molecules (e.g., inhibitors of target enzymes,competitors for binding of a natural ligand to its receptor, agonists orantagonists for receptor-mediated intracellular processes, to name afew), toxicity evaluation or bioavailability assessment, etc.

In certain exemplary embodiments, compounds of this invention wereassayed for their ability to:

-   -   inhibit DNA synthesis (e.g., DNA replication);    -   exhibit genistein suppressor activity;    -   exhibit Eg5 inhibitory activity;

Thus, in one aspect, compounds of this invention which are of particularinterest include those which:

-   -   inhibit DNA synthesis (e.g., DNA replication);    -   have genistein suppressor activity;    -   exhibit Eg5 inhibitory activity;    -   exhibit cytotoxic or growth inhibitory effect on cancer cell        lines maintained in vitro or in animal studies using a        scientifically acceptable cancer cell xenograft model;    -   exhibit a therapeutic profile (e.g., optimum safety and curative        effect) that is comparable or superior to existing        chemotherapeutic agents.

As discussed above, the compounds of the invention may be assayed forany of a variety of biological activities (e.g., in high-throughputscreening assays). For example, the library members may be arrayedaccording to the method described in patent application Ser. No.09/567,910, filed May 10, 2000, which is incorporated herein byreference in its entirety, and screened for detecting binding and/oractivation events occurring between members in the inventive library andbiological macromolecules of interest (e.g., for identifying smallmolecule partners (library members) for biological macromolecules ofinterest). The partners may be compounds that bind to particularmacromolecules of interest and are capable of activating or inhibitingthe biological macromolecules of interest. As discussed above, in oneaspect, the present invention provides methods, referred to herein as“small molecule printing”, for the generation of high density arrays andthe resulting compositions. According to the method of the presentinvention, a collection of chemical compounds, or one type of compound,can be “printed” onto a support to generate extremely high densityarrays. In certain embodiments, one or more library members may bearrayed by (1) providing a solid support, wherein the solid support isfunctionalized with a desired chemical moiety capable of interactingwith a desired chemical compound to form an attachment; (2) providingone or more solutions of the library members to be attached to the solidsupport; and (3) delivering the one or more solutions of the librarymembers to the solid support, whereby an array of compounds is generatedand the array has a density of at least 1000 spots per cm². In certainexemplary embodiments, a silylation reaction can be employed to attachthe library members to a glass slide.

In certain embodiments, plain glass slides are derivatized to yieldsurfaces that are densely functionalized with silyl halides. Compoundscontaining hydroxyl groups (e.g., library members) can then be providedand contacted with the functionalized glass surface. The hydroxylcontaining compounds readily attach to the surface through thesilicon-oxygen bond formed by nucleophilic substitution on the silylhalide. In a preferred embodiment, the silyl halide is silyl chloride,bromide, or iodide. In other preferred embodiments, leaving groups onthe silicon such as mesylate and tosylate are used rather than halides.Preferably, the hydroxyl groups of the compounds to be attached areunhindered (e.g., primary alcohols). See, for example, Hergenrother etal., J. Am. Chem. Soc., 122:7849-7850, 2000, which is incorporatedherein by reference in its entirety.

In certain embodiments, assaying the library members may be accomplishedby (1) arraying the library members, as described above, with a densityof at least 1000 spots per cm²; (2) contacting the array with one ormore types of biological macromolecules of interest; and (3) determiningthe interaction of specific small molecule-biological macromoleculepartners.

It will also be appreciated that the arrays of compounds may be utilizedin a variety of ways to enable detection of interactions between librarymembers and biological macromolecules. In one particularly preferredembodiment, an array of different types of chemical compounds attachedto the surface is utilized and is contacted by one or a few types ofbiological macromolecules to determine which compounds are capable ofinteracting with the specific biological macromolecule(s). As one ofordinary skill in the art will realize, if more than one type ofcompound is utilized, it is desirable to utilize a method for encodingeach of the specific compounds so that a compound having a specificinteraction can be identified. Specific encoding techniques have beenrecently reviewed and these techniques, as well as other equivalent orimproved techniques, can be utilized in the present invention (see,Czarnik, A. W. Current Opinion in Chemical Biology 1997, 1, 60; which isincorporated herein by reference in its entirety). Alternatively thearrays of the present invention may comprise one type of chemicalcompound and a library of biological macromolecules may be contactedwith this array to determine the ability of this one type of chemicalcompound to interact with a variety of biological macromolecules.

As one of ordinary skill in the art will realize, the biologicalmacromolecule of interest may comprise any biomolecule. In preferredembodiments, the biological macromolecule of interest comprises aprotein, and more preferably the array is contacted with a library ofrecombinant proteins of interest. In yet another preferred embodiment,the biological molecules of interest are provided in the form of celllysates such as, for example, those of tumor-associated cells. As willbe appreciated by one of ordinary skill in the art, these proteins maycomprise purified proteins, pools of purified proteins, and complexmixtures such as cell lysates, and fractions thereof, to name a few.Examples of particularly preferred biological macromolecules to studyinclude, but are not limited to those involved in signal transduction,dimerization, gene regulation, cell cycle and cell cycle checkpoints,and DNA damage checkpoints. Furthermore, the ability to constructlibraries of expressed proteins from any organism or tissue of interestwill lead to large arrays of recombinant proteins. The compounds ofinterest may be capable of either inactivating or activating thefunction of the particular biomolecule of interest.

In certain exemplary embodiments, the inventive library may be screenedto identify those library members capable of exerting an effect on anintracellular biological or chemical process. For a detailed descriptionof the screening method, see U.S. patent application Ser. No. 09/361,576and PCT Patent Application No.: US99/17046, each of which isincorporated herein by reference in its entirety. In one aspect, themethod encompasses screening chemical compounds for their effects onchemical and/or biological systems by detecting the presence or amountof a component present or produced by the system, which component actsas a marker for the chemical or biological process of interest. Often,detection of the presence or amount of such a biological component willreveal a perturbation in an underlying biological process. For example,the biological component may be a component or product of a cellsignaling pathway, so that detection of the component allows theidentification of test compounds that perturb the pathway. In certainembodiments, whole cells may be arrayed on a suitable solid support andone or more library members may be contacted with the arrayed cellsunder conditions suitable for at least one of the test compounds toexert an effect on an intracellular biological or chemical process. Aligand may then be contacted with said cells in each reaction vesselunder conditions suitable for said ligand to associate intracellularlywith at least one biological component whose presence or amount isindicative of said biological or chemical process. Finally, the presenceor amount of the ligand associated with said biological component may bemeasured with a suitable detection method. Preferably, the biologicalcomponent is detected by means of its interaction with a binding partnerligand. Preferably, the binding is specific. In certain preferredembodiments, the binding partner ligand is an antibody. In certainembodiments, the library may be screened to identify compounds thateffect changes in a variety of different cellular processes, including,for example, protein concentration, protein phosphorylation,methylation, acetylation, lipidation, isoprenylation, ubiquitination,second messenger concentration, and the rate or extent of DNA synthesis.

In certain embodiments, compounds of the invention inhibit BrdUincorporation in cells. BrdU (5-bromodeoxyuridine) is a thymidine analogin which the methyl group at the 5-position is replaced with bromine(FIG. 2 a). This analog is efficiently incorporated into DNA during DNAreplication, and can be detected with an antibody raised specificallyagainst this modified form. By detecting the incorporation of a naturalnucleotide or non-natural nucleotide, it is possible to determine growthand viability of a cell or collection of cells. DNA synthesis in a cellis an indicator of growth and viability. Therefore, compounds which mayaffect cell growth, the cell cycle, and viability of cells may beassayed using BrdU as a signaling cellular component. In certainexemplary embodiments, inventive compounds are useful for the treatmentof disorders associated with abnormal cell growth or cell proliferation(e.g., cancer).

In certain other embodiments, compounds of the invention exhibit Eg5inhibitory activity. Eg5 is a kinesin-related motor essential forbipolar spindle formation in vivo. Mitotic kinesins are enzymesessential for assembly and function of the mitotic spindle, but are notgenerally part of other microtubule structures, such as nerve processes.Mitotic kinesins play essential roles during all phases of mitosis.These enzymes are “molecular motors” that translate energy released byhydrolysis of ATP into mechanical force which drives the directionalmovement of cellular cargoes along microtubules. The catalytic domainsufficient for this task is a compact structure of approximately 340amino acids. During mitosis, kinesins organize microtubules into thebipolar structure that is the mitotic spindle. Kinesins mediate movementof chromosomes along spindle microtubules, as well as structural changesin the mitotic spindle associated with specific phases of mitosis.Experimental perturbation of mitotic kinesin function causesmalformation or dysfunction of the mitotic spindle, frequently resultingin cell cycle arrest. From both the biological and enzymaticperspectives, these enzymes are attractive targets for the discovery anddevelopment of novel anti-mitotic chemotherapeutics.

In yet other embodiments, compounds of the invention exhibit genisteinsuppressor activity. Genistein (4′,5,7-trihydroxyisoflavone) is abroad-spectrum protein tyrosine kinase inhibitor that has been shown tohave growth inhibitory effects against several cancers both in vitro andin vivo.

Pharmaceutical Compositions

In another aspect, this invention also provides pharmaceuticalpreparations comprising at least one of the compounds as described aboveand herein, optionally, though typically in combination with apharmaceutically acceptable carrier. In certain embodiments, thecompounds are capable of inhibiting the growth of or killing cancercells. Thus, the present invention provides pharmaceutical compositionsfor treating cancer, preferably for preventing the recurrence of cancer,comprising a compound of the present invention disclosed herein, as anactive ingredient, optionally, though typically in combination with apharmaceutically acceptable carrier.

As detailed herein, several of the inventive compositions have beendetermined to have a wide range of biological activities (e.g.,inhibition of Eg5 ATPase activity, FKBP12 binding, inhibition of DNAreplication, genistein suppressor activity). Thus, in another aspect ofthe present invention, pharmaceutical compositions are provided, whereinthese compositions include a compound that is useful in treating a“physiological condition,” defined herein as any biological orbiochemical process that affects the health of an individual, and apharmaceutically acceptable carrier. It will be appreciated that theinventive pharmaceutical compositions encompasses each of thosecompounds identified that inhibit or activate any physiological process.

It will also be appreciated that certain of the compounds of the presentinvention can exist in free form for treatment, or where appropriate, asa pharmaceutically acceptable derivative thereof. According to thepresent invention, a pharmaceutically acceptable derivative includes,but is not limited to, pharmaceutically acceptable salts, esters, saltsof such esters, or any other adduct or derivative which uponadministration to a patient in need is capable of providing, directly orindirectly, a compound as otherwise described herein, or a metabolite orresidue thereof, e.g., a prodrug.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge, etal. describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 66: 1-19 (1977), incorporated herein byreference. The salts can be prepared in situ during the final isolationand purification of the compounds of the invention, or separately byreacting the free base function with a suitable organic acid. Examplesof pharmaceutically acceptable, nontoxic acid addition salts are saltsof an amino group formed with inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid orwith organic acids such as acetic acid, oxalic acid, maleic acid,tartaric acid, citric acid, succinic acid or malonic acid or by usingother methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, magnesium, and the like.Further pharmaceutically acceptable salts include, when appropriate,nontoxic ammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, lower alkyl sulfonate and aryl sulfonate.

Additionally, as used herein, the term “pharmaceutically acceptableester” refers to esters which hydrolyze in vivo and include those thatbreak down readily in the human body to leave the parent compound or asalt thereof. Suitable ester groups include, for example, those derivedfrom pharmaceutically acceptable aliphatic carboxylic acids,particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, inwhich each alkyl or alkenyl moiety advantageously has not more than 6carbon atoms. Examples of particular esters includes formates, acetates,propionates, butyrates, acrylates and ethylsuccinates.

Furthermore, the term “pharmaceutically acceptable prodrugs” as usedherein refers to those prodrugs of the compounds of the presentinvention which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswith undue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use, as well as the zwitterionic forms, where possible,of the compounds of the invention. The term “prodrug” refers tocompounds that are rapidly transformed in vivo to yield the parentcompound of the above formula, for example by hydrolysis in blood. Athorough discussion is provided in T. Higuchi and V. Stella, Pro-drugsas Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series, andin Edward B. Roche, ed., Bioreversible Carriers in Drug Design, AmericanPharmaceutical Association and Pergamon Press, 1987, both of which areincorporated herein by reference.

As described above, the pharmaceutical compositions of the presentinvention additionally comprise a pharmaceutically acceptable carrier,which, as used herein, includes any and all solvents, diluents, or otherliquid vehicle, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, solidbinders, lubricants and the like, as suited to the particular dosageform desired. Remington's Pharmaceutical Sciences, Fifteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1975) discloses variouscarriers used in formulating pharmaceutical compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutical composition, its use is contemplatedto be within the scope of this invention. Some examples of materialswhich can serve as pharmaceutically acceptable carriers include, but arenot limited to, sugars such as lactose, glucose and sucrose; starchessuch as corn starch and potato starch; cellulose and its derivativessuch as sodium carboxymethyl cellulose, ethyl cellulose and celluloseacetate; powdered tragacanth; malt; gelatin; talc; excipients such ascocoa butter and suppository waxes; oils such as peanut oil, cottonseedoil; safflower oil; sesame oil; olive oil; corn oil and soybean oil;glycols; such a propylene glycol; esters such as ethyl oleate and ethyllaurate; agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol, and phosphate buffer solutions, as well asother non-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

Uses of Compounds and Pharmaceutical Compositions

In yet another aspect, according to the methods of treatment of thepresent invention, physiological conditions are treated or prevented ina subject such as a human, lower mammal, or other organism, byadministering to the patient a therapeutically effective amount of aninventive compound or pharmaceutical composition thereof, as describedin detail above, in such amounts and for such time as is necessary toachieve the desired result. In certain embodiments of the presentinvention a “therapeutically effective amount” of an inventive compoundor pharmaceutical composition is that amount effective for reducing thesymptoms associated with the physiological condition. In other preferredembodiments, a “therapeutically effective amount” of an inventivecompound or pharmaceutical composition is that amount effective foraffecting the secretory pathway of a cell. Other “therapeuticallyeffective amounts” include amounts effective for inhibiting the cellcycle, e.g., inhibiting the growth of cancer cells. Alternatively oradditionally, a “therapeutically effective amount” is an amount that iseffective for inhibiting or activating a physiological process ofinterest, wherein the physiological process is related to improving thehealth of the individual.

The compounds and compositions, according to the method of the presentinvention, may be administered using any amount and any route ofadministration effective for obtaining the physiological result. Thus,the expression “therapeutically effective amount,” as used herein,refers to a nontoxic but sufficient amount of an inventive compound toprovide the desired treatment. The exact amount required will vary fromsubject to subject, depending on the species, age, and general conditionof the subject, the severity of the physiological condition (e.g., aproliferative disorder or cancer), the particular compound, its mode ofadministration, and the like. The compounds of the invention arepreferably formulated in dosage unit form for ease of administration anduniformity of dosage. The expression “dosage unit form” as used hereinrefers to a physically discrete unit of compound appropriate for thepatient to be treated. It will be understood, however, that the totaldaily usage of the compounds and compositions of the present inventionwill be decided by the attending physician within the scope of soundmedical judgment. The specific therapeutically effective dose level forany particular patient or organism will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the activity of the specific compound employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and rateof excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificcompound employed; and like factors well known in the medical arts (seeGoodman and Gilman's, “The Pharmacological Basis of Therapeutics”, TenthEdition, A. Gilman, J. Hardman and L. Limbird, eds., McGraw-Hill Press,155-173, 2001, which is incorporated herein by reference in itsentirety).

Furthermore, after formulation with an appropriate pharmaceuticallyacceptable carrier in a desired dosage, the pharmaceutical compositionsof this invention can be administered to humans and other animalsorally, rectally, parenterally, intracistemally, intravaginally,intraperitoneally, topically (as by powders, ointments, or drops),bucally, as an oral or nasal spray, or the like, depending on theseverity of the condition being treated. In certain embodiments, thecompounds of the invention may be administered orally or parenterally atdosage levels of about 0.01 mg/kg to about 50 mg/kg and preferably fromabout 0.1 mg/kg to about 25 mg/kg, of patient body weight per day, oneor more times a day, to obtain the desired therapeutic effect.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle. Injectable depot forms are made by forming microencapsulematrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of drug to polymerand the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes. Solid compositions of asimilar type may also be employed as fillers in soft and hard-filledgelatin capsules using such excipients as lactose or milk sugar as wellas high molecular weight polyethylene glycols and the like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich can be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, and eye drops are also contemplatedas being within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms can be made by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

The invention further encompasses compounds and pharmaceuticalcompositions employed in combination therapies, that is, the compoundsand pharmaceutical compositions can be administered concurrently with,prior to, or subsequent to, one or more other desired therapeutics ormedical procedures The particular combination of therapies (therapeuticsor procedures) to employ in a combination regimen will take into accountcompatibility of the desired therapeutics and/or procedures and thedesired therapeutic effect to be achieved. It will also be appreciatedthat the therapies employed may achieve a desired effect for the samedisorder, or they may achieve different effects.

For example, other compounds that may be used in combination with thecompounds that can be provided using the structural information of thepresent invention. For example, if the inventive compound is achemotherapeutic agent, a second or third chemotherapeutic agent, suchas cisplatin, may be administered with the inventive compound to achievethe benefit of their combined effects. As but another example, if thecompound were to treat or prevent a reproductive disorder, the inventivecompound may be administered with a hormone, such as testosterone orestrogen. For a more comprehensive discussion regarding physiologicalconditions, symptoms and treatment, see The Merck Manual, SeventeenthEd. 1999, the entire contents of which are hereby incorporated byreference.

In certain embodiments, the pharmaceutical compositions of the presentinvention may further comprise other therapeutically active ingredients(e.g., chemotherapeutic and/or palliative). For purposes of theinvention, the term “Palliative” refers to treatment that is focused onthe relief of symptoms of a disease and/or side effects of a therapeuticregimen, but is not curative. For example, palliative treatmentencompasses painkillers, antinausea medications and anti-sickness drugs.In addition, chemotherapy, radiotherapy and surgery can all be usedpalliatively (that is, to reduce symptoms without going for cure; e.g.,for shrinking tumors and reducing pressure, bleeding, pain and othersymptoms of cancer).

In yet another aspect, the present invention also provides apharmaceutical pack or kit comprising one or more containers filled withone or more of the ingredients of the pharmaceutical compositions of theinvention, and in certain embodiments, includes an additional approvedtherapeutic agent for use as a combination therapy. Optionallyassociated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceutical products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration.

Methods of Treatment

In another embodiment, the compounds of the present invention, e.g.,compounds having cell cycle inhibitory activity or kinesin (e.g., Eg5)inhibitory activity, may be administered to a subject to treat orprevent cancer including, but are not limited to, adenocarcinoma,leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma,and, in particular, cancers of the adrenal gland, bladder, bone, bonemarrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinaltract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid,penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid,and uterus, to name a few.

In another embodiment, the compounds of the present invention, e.g.,inhibitors of Eg5, may be administered to a subject to prevent or treata proliferative disorder. Such disorders may include, but are notlimited to, disorders or prolactin production; infertility includingtubal disease, ovulatory defects, and endometriosis; disruptions of theestrous cycle, disruptions of the menstrual cycle, polycystic ovarysyndrome, ovarian hyperstimulation syndrome, endometrial and ovariantumors, autoimmune disorders, ectopic pregnancy, and teratogenesis;cancer of the breast, fibrocystic breast disease, and galactorrhea; anddisruptions of spermatogenesis, abnormal sperm physiology, cancer of thetestis, cancer of the prostate, benign prostatic hyperplasia,prostatitis, carcinoma of the male breast, and gynecomastia.

As discussed above, the methods and compositions herein are not limitedto cancer. Disease states which can be treated by the methods andcompositions provided herein include, but are not limited to, cancer(further discussed below), restenosis, autoimmune disease, arthritis,graft rejection, inflammatory bowel disease, proliferation induced aftermedical procedures, including, but not limited to, surgery, angioplasty,and the like. It is appreciated that in some cases the cells may not bein a hyper or hypo proliferation state (abnormal state) and stillrequire treatment. For example, during wound healing, the cells may beproliferating “normally”, but proliferation enhancement may be desired.Similarly, as discussed above, in the agriculture arena, cells may be ina “normal” state, but proliferation modulation may be desired to enhancea crop by directly enhancing growth of a crop, or by inhibiting thegrowth of a plant or organism which adversely affects the crop. Thus, inone embodiment, the invention herein includes application to cells orindividuals afflicted or impending affliction with any one of thesedisorders or states.

In certain embodiments, the compositions and methods provided herein areuseful for the treatment of cancer including solid tumors such as skin,breast, brain, cervical carcinomas, testicular carcinomas, etc. Moreparticularly, cancers that may be treated by the compositions andmethods of the invention include, but are not limited to: Cardiac:sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma),myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogeniccarcinoma (squamous cell, undifferentiated small cell, undifferentiatedlarge cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchialadenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma;Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma,leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma,glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel(adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma); Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor[nephroblastoma], lymphoma, leukemia), bladder and urethra (squamouscell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver:hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastom,angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenicsarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma,chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cellsarcoma), multiple myeloma, malignant giant cell tumor chordoma,osteochronfroma (osteocartilaginous exostoses), benign chondroma,chondroblastoma, chondromyxofibroma, osteoid osteoma and giant celltumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma,osteitis deformans), meninges (rheningioma, meningiosarcoma,gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma,germinoma [pinealoma, glioblastoma multiform, oligodendroglioma,schwannoma, retinoblastoma, congenital tumors), spinal cordneurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus(endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervicaldysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma,mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecalcell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignantteratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma,adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma,squamous cell carcinoma, botryoid sarcoma [embryonal rhabdomyosarcoma],fallopian tubes (carcinoma); Hematoloaic: blood (myeloid leukemia [acuteand chronic], acute lymphoblastic leukemia, chronic lymphocyticleukemia, myeloproliferative diseases, multiple myeloma, myelodysplasticsyndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignantlymphoma]; Skin: malignant melanoma, basal cell carcinoma, squamous cellcarcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma,dermatofibroma, keloids, psoriasis; and Adrenal glands: neuroblastoma.The cancer can be solid tumors or metastatic. Thus, the term “cancerouscell” as provided herein, includes a cell afflicted by any one of theabove identified conditions.

Those skilled in the art will appreciate that the invention is by nomeans limited to the treatment of the above disorders, but can be usedto treat any disorder that may be identified by a practicing physicianand which symptoms may be decreased by the compounds of the invention.

In another aspect, diagnostic assays are provided herein. In oneembodiment, the cellular proliferation sequences are used in thediagnostic assays. This can be done on an individual gene orcorresponding polypeptide level. In a preferred embodiment, theexpression profiles are used, preferably in conjunction with highthroughput screening techniques to allow monitoring for expressionprofile genes and/or corresponding polypeptides. In a preferredembodiment, in situ hybridization of labeled cellular proliferationnucleic acid probes to tissue arrays is done. For example, arrays oftissue samples, including cellular proliferation tissue in variousstates and or time points and/or normal tissue, are made. In situhybridization as is known in the art can then be done. It is understoodthat conventional antibody and protein localization methods can also beused in diagnostic assays herein.

EQUIVALENTS

The representative examples which follow are intended to help illustratethe invention, and are not intended to, nor should they be construed to,limit the scope of the invention. Indeed, various modifications of theinvention and many further embodiments thereof, in addition to thoseshown and described herein, will become apparent to those skilled in theart from the full contents of this document, including the exampleswhich follow and the references to the scientific and patent literaturecited herein. It should further be appreciated that the contents ofthose cited references are incorporated herein by reference to helpillustrate the state of the art. The following examples containimportant additional information, exemplification and guidance which canbe adapted to the practice of this invention in its various embodimentsand the equivalents thereof.

EXEMPLIFICATION Example 1 Synthesis of Inventive Compounds

I. Description of Synthetic Methods

Small molecules have been used to explore many facets of biology forover a century. However, research in biology is not routinely performedusing this approach, in the way that it is with biochemical, genetic,and increasingly, genomic approaches. Several problems limit the use ofthe former approach. Arguably, the primary one is the lack of routineaccess to structurally complex and diverse small molecules that can beused to modulate biological systems. There are examples of simple,achiral modulators of biological systems, notably “drug-like” molecules,though in these cases the smaller size and complexity of the specieshave more to do with delivery and pharmacokinetic parameters than withaffinity and selectivity for a protein target. Without wishing to bebound to any particular theory, we propose that structurally complex anddiverse collections of “natural product-like”, rather than “drug-like”molecules will be better suited as biological probes. Diversity-orientedorganic synthesis, especially when coupled with an economical andefficient technology platform, offers the means to change thissituation, as it aims to synthesize complex and diverse small moleculesefficiently (S. L. Schreiber, Science 2000, 287, 1964-1969).Diversity-oriented synthesis represents a versatile tool to chemicalgenetics, which aims to explore biology with small molecules in asystematic way (See for example, (a) T. J. Mitchison, Chem. Biol. 1994,1, 3-6; (b) S. L. Schreiber, Bioorg. Med. Chem. 1998, 6, 1127-1152; (c)http://www-schreiber.chem.harvard.edu; and http://iccb.med.harvard.edu).

Though enantioselective catalysis is often used in target-orientedsynthesis, it is still relatively under explored in diversity-orientedsynthesis (See, for example, a) D. S. Tan, M. A. Foley, M. D. Shair, S.L. Schreiber, J. Am. Chem. Soc. 1998, 120, 8565-8566; b) D. S. Tan, M.A. Foley, B. R. Stockwell, M. D. Shair, S. L. Schreiber, J. Am. Chem.Soc. 1999, 121, 9073-9087; c) D. Lee, J. K. Sello, S. L. Schreiber, J.Am. Chem. Soc. 1999, 121, 10648-10649; d) D. R. Spring, S. Krishnan, S.L. Schreiber, J. Am. Chem. Soc. 2000, 122, 5656-5657; e) S. M. Sternson,J. B. Louca, J. C. Wong, S. L. Schreiber, J. Am. Chem. Soc. 2001, 123,1740-1747).

For other approaches to asymmetric diversity synthesis, see: a) J. S.Panek, B. Zhu, J. Am. Chem. Soc. 1997, 119, 12022-12023; b) D. A. Annis,O. Helluin, E. N. Jacobsen, Angew. Chem. 1998, 110, 2010-2012; Angew.Chem., Int. Ed. Engl. 1998, 37, 1907-1909; c) M. Reggelin, V. Brenig, R.Welcker, Tetrahedron Lett. 1998, 39, 4801-4804; d) N. Zou, B. Jiang, J.Comb. Chem. 1999, 2, 6-7; e) S. Henessian, J. Ma, W. Wang, TetrahedronLett. 1999, 40, 4631; f) I. Paterson, M. Donghi, K. Gerlach, Angew.Chem., Int. Ed. 2000, 39, 3315-3319.

In certain embodiments, reactions catalyzed by bis(oxazoline) metalLewis acid complexes were explored because of their high efficiency,selectivity, and broad substrate tolerance (See, a) J. S. Johnson, D. A.Evans, Acc. Chem. Res. 2000, 33, 325-335; b) K. A. Jorgensen, M.Johannsen, S. Yao, H. Audrain, J. Thorhauge, Acc. Chem. Res. 1999, 32,605-613). In certain exemplary embodiments, inverse electron demandheterocycloadditions of vinyl ethers and β,χ-unsaturated ketoesters(FIG. 2) were investigated. See, a) D. A. Evans, J. S. Johnson, E. J.Olhava, J. Am. Chem. Soc. 2000, 122, 1635-1649; b) D. A. Evans, E. J.Olhava, J. S. Johnson, J. M. Janey, Angew. Chem. 1998, 110, 3554-3557;Angew. Chem. Int. Ed. 1998, 37, 3372-3375; c) J. Thorhauge, M.Johannsen, K. A. Jorgensen, Angew. Chem. 1998, 110, 2543-2546; Angew.Chem. Int. Ed. 1998, 37, 2404-2406.

An account of related cycloadditions on solid support has been described(S. Leconte, G. Dujardin, E. Brown, Eur. J. Org. Chem. 2000, 639-643.For a related heterocycloaddition on solid support see: F. Tietze, T.Hippe, A. Steinmetz, Synlett 1996, 1043-1044. For a report of anasymmetric cycloaddition with external control on a solid support, seereference 5d); however, the reported reactions were performed in thepresence of achiral catalysts and with the heterodiene bound to thepolystyrene (PS) solid support through the ester. In certainembodiments, this mode of cycloaddition was initially investigated andfound to be highly selective when using the enantiomerically purecatalysts (S)- or (R)-1 [(a) D. A. Evans, J. S. Johnson, E. J. Olhava,J. Am. Chem. Soc. 2000, 122, 1635-1649; b) D. A. Evans, E. J. Olhava, J.S. Johnson, J. M. Janey, Angew. Chem. 1998, 110, 3554-3557; Angew. Chem.Int. Ed. 1998, 37, 3372-3375; c) J. Thorhauge, M. Johannsen, K. A.Jorgensen, Angew. Chem. 1998, 110, 2543-2546; Angew. Chem. Int. Ed 1998,37, 2404-2406].

In certain exemplary embodiments, support-bound vinyl ethers were used,that were linked to the macrobead through either carbon or oxygen. Thisapproach was found to be more effective with regard to effectivefunctionalization of the cycloadduct.

Described herein is an application of this asymmetric cycloadditionreaction to the synthesis of dihydropyrancarboxamides on high capacity,500-600 μm PS macrobeads in a one bead-one stock solution technologyplatform. The diversity pathway explored resulted in the highlydiastereo- and enantioselective synthesis of 4320 encoded smallmolecules [See (a) M. H. J. Ohlmeyer, R. N. Swanson, L. W. Dillard, J.C. Reader, G. Asouline, R. Kobayashi, M. Wigler, W. C. Still, Proc.Natl. Acad. Sci. USA. 1993, 90, 10922-10926; (b) H. P. Nestler, P. A.Bartlett, W. C. Still J. Org. Chem. 1994, 59, 4723-4724; and (c) H. E.Blackwell, L. Pérez, S. L. Schreiber, Angew. Chem. Int. Ed. 2001,40(18), 3421-3425], which were arrayed as 5 mM stock solutions fromindividual beads, each containing predominantly a singledihydropyrancarboxamide. These stock solutions permit many phenotypicand proteomic assays to be performed. For a description of a fullyautomated procedure for deriving and arraying stock solutions from thedihydropyrancarboxamide-containing macrobead, see Paul A. Clemons etal., “A one-bead, one-stock solution approach to chemical genetics: Part2”; Chemistry & Biology, 2001, 8:1183-1195.

Collections of vinyl ethers and unsaturated ketoesters were firstsynthesized and were used as candidate partners for the cycloadditionreaction. Depicted below are the vinyl ethers that were synthesized forthe study:

Depicted below are the unsaturated ketoesters that were synthesized forthe study:

One of ordinary skill in the art will recognize that other vinyl etherand/or unsaturated ketoester building blocks may be used to practice theinvention, leading to the preparation of dihydropyrancarboxamides otherthan those disclosed in the Exemplification herein, without departingfrom the scope of the invention.

In certain embodiments, each of the vinyl ethers BB1A-N was loaded ontopools of PS macrobeads via the in situ generated silyl triflate 3 asdepicted in Scheme 2. The support-bound vinyl ethers were then treatedwith heterodienes (either BB2-B, R=phenyl or BB2-E, R=4-piperonyl) (3equiv) in tetrahydrofuran (THF) in the presence of 20 mol % of the t-BuBOX—Cu(OTf)₂ complex ((S)- or (R)-1) and 4 Å molecular sieves to providesupport-bound cycloadducts (not shown) [Note: After surveying severalloading/ligand/metal/solvent combinations, 20 mol % of 1 in THF wasfound to provide the best combination of kinetics and selectivity]. Bothenantiomers of the ligand were used in separate reactions to obtain aduplicate result and to detect potential matched/mismatched pairs whenchiral starting materials were used. After washing and drying steps,each of the cycloadducts was cleaved from the silyl ether linker withhydrogen fluoride-pyridine (HF-py) and analyzed for purity using ¹H NMRspectroscopy and mass spectrometry (LC-MS).

These studies showed that support-bound vinyl ethers with amino or amidofunctionality led to low conversion, and the support-bound form ofbis(vinyl ether) BB1-N underwent a single, rather than the desireddouble, cycloaddition, even when a stoichiometric amount of the coppercomplex was used. The chiral vinyl ether derived from threitol(support-bound form of BB1-I) reacted efficiently with only the(S)-enantiomer of the catalyst, suggesting that doublediastereoselection was taking place (see below). Upon treatment of thecorresponding PS macrobeads with HF-py, the remaining vinyl ethersstudied provided high purity cycloadducts 4-6 (Table 1). The ethenylethers BB1-A, B, G, H also yielded dihydropyrans 4 in high diastereo-and enantioselectivity (The configurations of the cycloadducts wereassigned by analogy. See D. A. Evans, J. S. Johnson, E. J. Olhava, J.Am. Chem. Soc. 2000, 122, 1635-1649; and J. Thorhauge, M. Johannsen, K.A. Jorgensen, Angew. Chem. 1998, 110, 2543-2546; Angew. Chem. Int. Ed.1998, 37, 2404-2406). Both configurations of substituted enol ethersBB1-C—F led to good to high diastereoselectivity of the tetrasubstituteddihydropyrans 5 and 6. Although previous results had shown highdiastereoselectivity with cyclic vinyl ethers (See D. A. Evans, J. S.Johnson, E. J. Olhava, J. Am. Chem. Soc. 2000, 122, 1635-1649; and J.Thorhauge, M. Johannsen, K. A. Jorgensen, Angew. Chem. 1998, 110,2543-2546; Angew. Chem. Int. Ed. 1998, 37, 2404-2406), we found thatZ-configured enol ethers (BB1-D, F) provided only moderatediastereoselection, whereas the E-enol ethers (BB1-C, E) resulted inhigh levels of diastereoselection. Without wishing to be bound to anyparticular theory, we propose that the lower diastereoselectivity in theZ-enol ether cycloadditions may arise from an endo-exo switch in thetransition structure for cycloaddition. It was thought unlikely thatisomerization of the alkenyl ether and epimerization of the acetalcenter is/are responsible for the lower selectivity. TABLE 1 Asymmetriccycloadditions of resin-bound vinyl ethers BB1.^([i])

purity, BB1- BB2- product %^([ii]) dr^([iii]) er^([iv]) A E 7-A-E ≧95≧15/1 ≧49/1 B E 7-B-E ≧95 ≧15/1 ≧24/1 C B 9-C-B ≧95 ≧20/1 ≧49/1 D B8-D-B ≧95  ≧5/1 ≧30/1 E B 9-E-B ≧95 ≧20/1 ≧49/1 F B 8-D-B ≧95 ≧10/1≧30/1 G E 7-G-E ≧95 ≧30/1 ≧49/1 H E 7-G-E ≧95 ≧20/1 ≧24/1^([i])Reactions were performed with 20 mol % of (S)-1 or (R)-1; theresults presented are an average of the two runs.^([ii])Estimated based on ¹H NMR analysis and HPLC-ESIMS.^([iii])Determined by ¹H NMR analysis and/or CSP HPLC or CSP SFC.^([iv])Determined by CSP HPLC or CSP SFC.

We next turned our attention to the substitution on the heterodienepartner. In most instances, treatment of the support-bound vinyl etherBB1-H with a variety of hetereodienes under the previous conditionsagain led to highly pure cycloadducts following HF-py cleavage from thePS macrobeads (Table 2), though again amine functionality (BB2-K) wasincompatible. Similar to the case above with the threitol-derived vinylether, only the (S)-enantiomer of the catalyst efficiently providedcycloadduct with the mannose-derived heterodiene BB2-L. Overall, tenheterodienes (BB2-A-J) resulted in somewhat variable, but uniformly highdiastereo- and enantioselectivities and high purities based on ¹H NMRspectroscopy and LC-MS analyses. These building blocks were chosen forsubsequent incorporation into the library synthesis. TABLE 2 Asymmetriccycloadditions of resin-bound vinyl ether 4-H with heterodienes.^([i])

purity, BB2- products %^([ii]) dr^([iii]) er^([iv]) A 10-H-A ≧95 ≧16/1≧16/1 B 10-H-B ≧95 ≧20/1 ≧24/1 C 10-H-C ≧95  ≧9/1 ≧24/1 D 10-H-D ≧95 ≧9/1  ≧9/1 E 10-H-E ≧95 ≧20/1 ≧24/1 F 10-H-F ≧95 ≧25/1 ≧24/1 G 10-H-G≧95  ≧9/1 ≧49/1 H 10-H-H ≧95 ≧15/1 ≧24/1 I 10-H-I ≧95 ≧12/1 ≧49/1 J10-H-J ≧95  ≧9/1 ≧49/1^([i])Reactions were performed with 20 mol % of (S)-1 or (R)-1; theresults presented are an average of the two runs.^([ii])Estimated based on ¹H NMR analysis and HPLC-ESIMS.^([iii])Determined by ¹H NMR analysis and/or CSP HPLC or CSP SFC.^([iv])Determined by CSP HPLC or CSP SFC.

Further functionalization of the cycloadducts was then pursued.Conversion of the support-bound cycloadduct 7-H-E, upon treatment with(PPh₃)₄Pd and thiosalicylic acid, to the corresponding acid 11-H-E wasachieved in high purity (Scheme 2). Treatment of the support-bound acid11-H-E with 20 equiv of benzylamine, PyBOP, and diisopropylethylamine in3:1 CH₂Cl₂:DMF led to the desired benzylamide. These conditions wereapplied to a diverse collection of amines and support-bound acid 11-H-Eto select 25 amines for use in the library synthesis:

These pathway development studies were necessary to select the reactionsand building blocks for a library realization that would result insingle compound stock solutions from individual macrobeads. The librarysynthesis was initiated with sufficient PS macrobeads (13,000) toproduce, on average, three beads containing each theoretical compound.The chosen vinyl ethers were attached to the supports, and following theinitial cycloaddition step, the two enantiomeric sets of cycloadductswere not pooled. (Each set includes cycloadduct attached to either theC1 oxygen or C2 carbon of the dihydropyran ring.) Instead, the two setswere carried through the remaining steps in parallel in order to providean independent means (when coupled to mass spectrometry) to assess theability of tags to infer the absolute configuration of library members(See Scheme 3). The supports were not repooled following the amidecoupling, thereby reducing the number of chemical encoding steps towhich the macrobeads were subjected and simplifying the decoding oflibrary members. In the end, 54 separate portions of macrobeads wereproduced (50 portions containing dihydropyrancarboxamides, 2 containingdihydropyrancarboxylic acids, and 2 containing dihydropyrancarboxylicesters), each containing, theoretically, three copies of 80 compoundsfor a total of 4320 distinct, spatially-segregated, andstereochemically-defined dihydropyran derivatives.

In order to analyze the purity of members of the library, two macrobeadsfrom each of the above 54 pools were removed, arrayed, treated withHF-py, and fractions of the eluted products (10 μL of 5 mM stocksolutions) were assayed by LC-MS (See H. E. Blackwell, L. Pérez, S. L.Schreiber, Angew. Chem. Int. Ed. 2001, 40(18), 3421-3425). In summary,78 samples (72%) were ≧95% pure, 93 samples (86%) were ≧90% pure, 104samples (96%) were ≧75% pure, and the remaining 4 samples were ofroughly 50% purity. Direct structure determination by MS was successfulin 83/108 cases (e.g., for 25 of the 108 samples, the molecular ionobserved upon ionization corresponded to a fragment of the compound),and indirect structure inference by decoding of the chloroaromaticdiazoketone tags (See for example, H. P. Nestler, P. A. Bartlett, W. C.Still J. Org. Chem. 1994, 59, 4723-4724; and H. E. Blackwell, L. Pérez,S. L. Schreiber, Angew. Chem. Int. Ed. 2001, 40(18), 3421-3425) wassuccessful in 108/108 cases.

Although the Examples described herein disclose a synthesis of theinventive library using stereochemistry as a diversity element wherebyonly one of two potential diastereomers (for the unsubstituted vinylethers) was accessed, it is to be understood that other reagents (e.g.,catalyst) and/or reaction conditions may be used that would allow accessto the full set of diastereomers, without departing from the scope ofthe invention. For example, the present invention encompasses the use ofcatalyst systems with truly complete external control overenantioselectivity and diastereo-selectivity to allow access tostereoselective catalysis in diversity-oriented organic synthesis. Inaddition, in one aspect, the present invention discloses a novelapproach whereby asymmetric heterocycloaddition reaction is applied tosolid phase (To the best of our knowledge, this is the first report ofthe use of a sub-stoichiometric amount of chiral controller to perform acarbon-carbon bond forming reaction on solid phase). Additionally, inanother aspect, the present invention describes the generation ofspatially-segregated stock solutions from individual macrobeads, whichrenders the inventive compounds amenable to both phenotypic andprotein-binding assays. In addition, their common primary hydroxyl groupensures that every compound can be robotically arrayed onto a glassmicroscope slide for protein-binding assays [a) G. Macbeath, A. N.Koehler, S. L. Schreiber, J. Am. Chem. Soc. 1999, 121, 7967-7968; b) P.J. Hergenrother, K. M. Depew, S. L. Schreiber, J. Am. Chem. Soc. 2000,122, 7849-7850]. Indeed, small molecule microarrays of thedihydropyrancarboxamides have already been manufactured and screened,leading to the discovery of a small molecule that binds to a protein ofinterest.

II. Experimental Protocol

General Methods. Reagents were obtained from commercial sources and usedwithout purification. Reaction solvents (THF, DMF, CH₂Cl₂) were obtainedfrom J. T. Baker (HPLC grade) and purified by passage through twosolvent columns prior to use. The CH₂Cl₂ purification system wascomposed of one activated alumina (A-2) column and one supported copperredox catalyst (Q-5 reactant) column. The THF purification system wascomposed of two activated alumina (A-2) columns and the DMF purificationsystem is composed of two activated molecular sieve columns. [See: A. B.Pangborn, M. A. Giardello, R. H. Grubbs, R. K. Rosen, F. J. Timmers,Organometallics 1996, 15, 1518-1520.] Triethylamine,diisopropylethylamine, and 2,6-lutidine were distilled from CaH₂.Brominated polystyrene resin (Br—PS, 2 meq/g) was obtained from PolymerLabs, and functionalized with the silicon-based linker according to thereported protocol (See J. A. Tallarico, K. M. Depew, H. E. Pelish, N. J.Westwood, C. W. Lindsley, M. D. Shair, S. L. Schreiber, M. A. Foley, J.Comb. Chem. 2001, 3, 312-318).2,2′-Isopropylidenebis[(4S)-4-t-butyl-2-oxazoline] was purchased fromAldrich, while the enantiomer,2,2′-Isopropylidenebis[(4R)-4-t-butyl-2-oxazoline] was prepared aspreviously described starting for (R)-t-leucine (See D. A. Evans, G. S.Peterson, J. S. Johnson, D. M. Barnes, K. R. Campos, K. A. Woerpel, J.Org. Chem. 1998, 63, 4541-4544). Flash chromatography was performed onE. Merck 60 230-400 mesh silica gel. TLC was performed on 0.25 mm EMerck silica gel F₂₅₄ plates and visualized by UV, cerium ammoniummolybdate and/or I₂. NMR spectra were recorded on a Varian Mercury 400(400 MHz ¹H, 100 MHz ¹³C), Varian Unity 500 (500 MHz ¹H) or Varian Unity600 (600 MHz ¹H). Chemical shifts are quoted in ppm and reference to TMSor residual protonated solvent. Mass spectra were obtained on a JeolAX-505H or SX-102A mass spectrometer.

Solid Phase Reactions. All solid phase reactions were conducted in ovendried glass vials under an atmosphere of dry Ar, with mixing provide bya VWR Vortex Genie-2 vortexer. Resin washings were performed in 2 mLfritted polypropylene Bio-Spin® chromatography columns (BioRad) or 10 mLfritted polypropylene PD-10 columns (Pharmacia Biotech) with 360°rotation on a Barnstead-Thermolyne Labquake™ shaker. For cleavagereactions, resin samples were transferred to Eppendorf tubes and acleavage cocktail comprising 85/10/5 THF/py/HF-py was added and thesamples were vortexed for 1-2 h at rt. The samples were then treatedwith methoxytrimethylsilane and vortexed for an additional 30 min. Thesamples were then filtered through a pipette plugged with glass wool,the resin washed with additional THF and the filtrate concentrated.

(a) Synthesis of Vinyl Ethers

(E)-5-Ethoxy-pent-4-en-1-ol

5-Ethoxy-pent-4-yn-1-ol (1.28 g, 10.0 mmol, 1.0 equiv) in THF (2 mL) wasadded to a suspension LiAlH₄ (0.80 g, 21 mmol, 2.1 equiv) in THF (20 mL)and the resulting solution was stirred at rt for 16 h. At this time 0.8mL of H₂O was added dropwise, followed by 0.8 mL of 15% NaOH, then 2.4mL of H₂O. Eventually (30 min) white solids formed, the mixture wasfiltered and the solids were extensively washed with EtOAc. The filtrateand washings were combined, dried (Na₂SO₄), filtered, and the filtratewas concentrated to give and oil which was purified by columnchromatography (6/1 hexanes/EtOAc) to give 1.14 g (88%) of the vinylether BB1-C as a clear colorless oil with better than 19/1 E/Zselectivity (¹H NMR): ¹H NMR (500 MHz) 6.24 (d, J=12.7, 1H, HC (5));4.76 (dt, J=12.7, 7.3, 1H, HC (4)); 3.69 (q, J=6.8, 2H, H₂C (1′)); 3.65(q, J=5.3, 2H, H₂C (1′)); 2.01 (q, J=7.3, 2H, H₂C (3)); 1.60 (quint,J=7.3, 2H, H₂C (2)); 1.39 (t J=5.4, 1H, OH); 1.25 (t, J=6.8, 3H, H₃C(2′)); ¹³C NMR (100 MHz) 146.12; 103.26; 64.56; 61.99; 33.46; 24.08;14.85.

tert-Butyl-{2-[2-(2-chloro-ethoxy)-ethoxy]-ethoxy}-dimethyl-silane

Triethylamine (9.2 mL, 66 mmol, 1.2 equiv), DMAP (0.67 g, 5.5 mmol, 0.1equiv) and TBSCl (9.1 g, 60.5 mmol, 1.1 equiv) were dissolved in CH₂Cl₂(60 mL). The 2-chloro(ethoxyethoxy)ethanol (8.0 mL, 55.0 mmol, 1.0equiv) was added dropwise over 5 min, then the mixture was stirred at rtfor 1.5 h. The reaction mixture was poured into H₂O and extracted withEtOAc, the combined organic layers were washed with brine, dried(MgSO₄), filtered and concentrated to give a cloudy oil which was passedthrough a short column of silica gel eluting with 9/1 hexane/EtOAc togive 14.8 g (96%) of the silyl ether as a clear, colorless oil: ¹H NMR(500 MHz) 3.74 (q, J=5.4, 4H, H₂C (1″) and H₂C (2)); 3.64 (s, 4H, H₂C(1′) and H₂C (2′)); 3.60 (t, J=5.9, 2H, H₂CO); 3.54 (t, J=5.9, 2H,H₂CO)); 0.87 (s, 9H, H₃CCSi); 0.04 (s, 6H, H₃CSi) ¹³C NMR (100 MHz)δ2.67; 71.31; 70.68; 70.66; 42.59; 25.86; 18.30; -5.34.

2-[2-(4-Ethoxy-but-3-ynyloxy)-ethoxy]-ethanol

tert-Butyl-{2-[2-(2-chloro-ethoxy)-ethoxy]-ethoxy}-dimethyl-silane wasconverted to the corresponding iodide by refluxing with 5 equiv of NaIin 2-butanone for 6 h. This was then alkynylated and deprotected by thesame process described above to provide E-5-Ethoxy-pent-4-en-1-ol: ¹HNMR (500 MHz) 4.02 (q, J=6.8, 2H, H₂C (5″)); 3.74-3.70 (m, 2H, HCO);3.68-3.66 (m, 2H, HCO); 3.64-3.60 (m, 4H, HCO); 3.54 (t, J=7.3, 2H,H₂CO)); 2.49 (t, J=6.3, 1H, OH); 2.41 (t, J 7.3, 2H, H₂CO); 1.33 (t,J=7.3, 3H, H₃C (6″)); ¹³C NMR (100 MHz) 128.24; 89.89; 73.91; 72.47;70.56; 70.25; 70.14; 61.66; 18.38; 14.26; MS (CI, NH₃) 220 (M+NH₄); 186.

(Z)-5-Ethoxy-pent-4-en-1-ol

5-Ethoxy-pent-4-yn-1-ol (1.28 g, 10 mmol) and Lindlar catalyst (400 mg)were combined in EtOAc (20 mL) containing pyridine (1 mL). This mixturewas hydrogenated at rt under 1 atm of H₂ for 16 h, then filtered throughcelite and poured into H₂O. The organic later was washed with sat.CuSO₄, H₂O and brine, then dried (Na₂SO₄), filtered, and the filtrateconcentrated to give an oil which was purified by column chromatography(6/1 hexanes/EtOAc) to give 0.91 g (70%) of the vinyl ether BB1-D as aslightly yellow oil with better than 25/1Z/E selectivity (¹H NMR): ¹HNMR (500 MHz) 6.01 (d, J=6.4, 1H, HC (5)); 4.36 (q, J=7.8, 1H, HC (4));3.79 (q, J=7.3, 2H, H₂C (1′)); 3.63 (t, J=6.3, 2H, H₂C (1); 2.17 (q,J=7.8, 2H, H₂C (3)); 2.06 (br s, 1H, OH); 1.59 (quint, J=6.3, 2H, H₂C(2)); 1.25 (t, J=7.3, 3H, H₃C2′)); ¹³C NMR (100 MHz) 145.20; 105.45;67.55; 61.56; 31.83; 19.71; 15.18.

(E)-2-[2-(4-Ethoxy-but-3-enyloxy)-ethoxy]-ethanol

The procedure described above for the synthesis of BB1-C provided theZ-vinyl ether BB1-E: ¹H NMR (500 MHz) 6.29 (d, J=12.7, 1H, HC (4″));4.73 (dt, J=12.2, 7.8, 1H, HC (3″)); 3.76-3.64 (m, 6H, H₂CO); 3.62-3.59(m, 4H, H₂CO); 3.44 (t, J=6.8, 2H, H₂C (1″)); 2.21 (qd, J=7.3, 1.0, 2H,H₂C (2″)); 1.25 (t, J=7.3, 3H, H₃C (6″)); ¹³C NMR (100 MHz) 147.41;99.52; 72.44; 71.97; 70.25; 70.05; 64.44; 61.59; 28.13; 14.60; MS (CI,NH₃) 222 (M+NH₄); 176.

(Z)-2-[2-(4-Ethoxy-but-3-enyloxy)-ethoxy]-ethanol

The procedure described above for the synthesis of BB1-D provided theZ-vinyl ether BBL-F: ¹H NMR (400 MHz) 5.99 (dt, J=6.3, 1.5, 1H, HC(4″)); 4.36 (q, J=6.3, 1H, HC (3″)); 3.77 (q, J=6.8, 2H, H₂C (5″));3.74-3.70 (m, 2H, HCO); 3.68-3.66 (m, 2H, HCO); 3.62-3.58 (4H, m, H₂CO);3.48 (t, J=7.3, 2H, H₂C (1″)); 2.56 (br s, 1H, OH); 2.37 (qd, J=6.8,1.5, 2H, H₂C (2″)); 1.23 (t, J=7.3, 3H, H₃C (6″)); ¹³C NMR (100 MHz)146.01; 102.19; 72.45; 71.00; 70.43; 69.95; 67.53; 61.77; 24.50; 15.21;MS (CI, NH₃) 222 (M+NH₄); 176; 159.

(4-Vinyloxymethyl-phenyl)-methanol

Benzene-1,4-dimethanol (2.76 g, 20.0 mmol, 1.0 equiv) and Hg(OAc)₂ (1 g,3.0 mmol, 0.15 equiv) were heated to reflux in butyl vinyl ether (50 mL)for 30 min. The reaction mixture was cooled, poured into sat. NaHCO₃ andwas extracted with EtOAc. The organic extracts were combined, washedwith H₂O and brine, dried (Na₂SO₄), filtered and the filtrateconcentrated to give a paste, which contained both mono- and bis-vinylethers and starting diol. Column chromatography (4/1 hexanes/EtOAc)provided 1.07 g (33%) of the mono-vinyl ether BB1-G as an oil whichsolidified on standing: ¹H NMR (400 MHz) 7.39-7.34 (m, 4H, HAr); 6.65(dd, J=14.6, 7.0, 1H, HC (1′)); 4.76 (s, 2H, H₂C (5)); 4.70 (d, J=5.9,2H, H₂COH)); 4.30 (dd, J=14.6, 2.2, 1H, HC (2′)); 4.08 (dd, J=7.0, 2.2,1H, HC (2′)); 1.62 (t, J=5.9, 1H, OH); ¹³C NMR (100 MHz) 151.50; 140.57;136.13; 127.70; 127.04; 87.39; 69.75; 64.83; MS (CI, NH₃) 182 (M+NH₄);138; 100.

tert-Butyl-(3-iodo-propoxy)-dimethyl-silane

Triethylamine (8.5 mL, 61.0 mmol, 1.2 equiv), DMAP (0.610 g, 5.0 mmol,0.1 equiv) and TBSCl (8.4 g, 55.5 mmol, 1.1 equiv) were dissolved inCH₂Cl₂ (50 mL). 3-Iodopropanol (9.4 g, 50.5 mmol, 1 equiv) was added andthe mixture was allowed to stir at rt for 16 h. The cloudy mixture wasthen poured into H₂O and extracted with hexane. The combined organiclayers were washed with H₂O, sat. CuSO₄, H₂O and brine, then dried(Na₂SO₄), filtered and the filtrate was concentrated to give an oil. Thecrude oil was purified by passed through a short plug of silica gelusing 19/1 hexanes/EtOAc as eluent to give 13.9 g (92%) of the silylether as a clear, colorless oil: ¹H NMR (500 MHz) 3.67 (t, J=5.9, 2H,H₂C (1)); 3.28 (t, J=6.8, 2H, H₂C (3)); 1.99 (quint, J=5.9, 2H, H₂C(2)); 0.89 (s, 9H, H₃CCSi)); 0.07 (s, 6H, H₃CSi). ¹³C NMR (100 MHz)δ2.33; 36.14; 25.90; 18.27; 3.68; -5.33.

5-Ethoxy-pent-4-yn-1-ol

A solution of ethyl alkynyl ether (7.8 g of a 40% wt. soln. in hexanes,roughly 3.1 g alkynyl ether, 44 mmol, 1.2 equiv) in THF (80 mL) wascooled to −78° C. and nBuLi (16.1 mL of 2.5 M in hexane, 40.3 mmol, 1.1equiv) was added over 5 min. This solution was allowed to stir for 20min at −78° C., then HMPA (14.0 mL, 80.6 mmol, 2.2 equiv) was added andthe solution was stirred for a further 20 min, thentert-butyl-(3-iodo-propoxy)-dimethyl-silane (11.0 g, 36.6 mmol, 1.0equiv) was added over 1 min and the mixture was allowed to warm slowlyto rt and stir overnight (16 h). The crude reaction mixture was pouredinto H₂O and extracted with hexane. The organic layers were combined,washed with brine, dried (Na₂SO₄), filtered and the filtrate wasconcentrated to give a dark oil which was immediately dissolved in THF(35 mL). TBAF (1.0 M in THF, 38 mL, 38 mmol, 1.04 equiv) was then addedand the resulting solution was stirred at rt for 1 h, the then reactionmixture was poured into H₂O and extracted with EtOAc. The combinedextracts were washed with H₂O and brine, dried (Na₂SO₄), filtered andthe filtrate was concentrated to give a dark oil which waschromatographed (6/1 hexanes/EtOAc) to give 3.43 g (73%) of the alkynylether as a slightly yellow oil: ¹H NMR (500 MHz) 4.02 (q, J=7.3, 2H, H₂C(2′)); 3.73 (q, J=5.8, 2H, H₂C (1)); 2.24 (t, J=6.8, 2H, H₂C (3)); 1.70(quint, J=6.8, 2H, H₂C (2)); 1.65 (t, J=5.9, 1H, OH); 1.33 (t, J=7.3,3H, H₃C (2′)); ¹³C NMR (100 MHz) 128.20; 89.55; 73.83; 61.67; 32.11;14.22; 13.67.

N,N-Bis-(2-hydroxy-ethyl)-4-methoxy-benzenesulfonamide

Diethanolamine (8.40 g, 80 mmol, 1.50 equiv) was dissolved in CH₂Cl₂ (30mL) and pyridine (5.1 mL, 63.6 mmol, 1.2 equiv). A solution of4-methoxybenzenesulfonyl chloride (10.9 g, 53 mmol, 1.0 equiv) n CH₂Cl₂(30 mL) was added quickly and the resulting mixture was stirred at rtovernight, then poured into H₂O and extracted with EtOAc. The organiclayers were washed with 1M HCl and brine, dried (MgSO₄), filtered, andthe filtrate was concentrated to give an oil which was purified bycolumn chromatography (95/5 CH₂Cl₂/MeOH) to give 12 g (83%) of thesulfonamide as an oil which crystallized on standing: ¹H NMR (500 MHz)7.75 (d, J=8.8, 2H, HAr); 6.99 (d, J=8.8, 2H, HAr); 3.87 (a, 3H, H₃CO);3.86 (t, J=4.9, 4H, H₂C (1′); 3.42 (br s, 2H, OH); 3.26 (t, J=4.9, 4H,H₂C (2′)). ¹³C NMR (100 MHz) 163.05; 129.85; 129.37; 114.37; 62.25;55.61; 52.90.

N-(2-Hydroxy-ethyl)-4-methoxy-N-(2-vinyloxy-ethyl)-benzenesulfonamide

The general procedure above for the synthesis of monovinyl ether BB1-Gprovided 38% of the desired vinyl ether sulfonamide BB1-H: ¹H NMR (500MHz) 7.76 (d, J=8.8, 2H, HAr); 6.99 (d, J=8.8, 2H, HAr); 6.42 (dd,J=14.7, 6.8, 1H, HC (3″)); 4.23 (dd, J=14.6, 2.4, 1H, HC (4″)); 4.07(dd, J=6.8, 2.4, 1H, HC (4″)); 3.93 (t, J=5.4, 2H, H₂C (2″)); 3.87 (s,3H, H₃CO); 3.76 (q, J=5.4, 2H, H₂C (2′)); 3.42 (t, J=5.4, 2H, H₂C (1″));3.27 (t, J=5.4, 2H, H₂C (1′)); 2.75 (t, J=6.3, 1H, OH); ¹³C NMR (100MHz) 163.00; 150.83; 130.12; 129.35; 114.31; 87.79; 67.48; 61.26; 55.58;56.62; 48.87.

2-(Trimethylsilanyloxy)-acrylic acid allyl ester

Triethylamine (33 mL, 235 mmol, 1.1 equiv) was added to a solution ofTMSOTf (50 g, 225 mmol, 1.05 equiv) in benzene (225 mL) and theresulting solution was cooled to 0° C. Allyl pyruvate (27.4 g, 214 mmol,1.0 equiv) was added over 30 min and the resulting two phase mixture wasstirred at 0° C. for another 2 h, then poured into ice cold H₂O andextracted with hexane. The hexane extracts were washed with H₂O, sat.CuSO₄, H₂O and brine, then dried (Na₂SO₄), filtered, and the filtratewas concentrated to give 30.4 g (68%) of the silyl enol ether as ayellow oil which was used without further purification: ¹H NMR (500 MHz)5.86 (ddd, J=171, 10.7, 1.4, 1H, HC (2′)); 5.55 (d, J=1.0, 1H, HC (4));5.35 (dd, J=17.1, 1.3, 1H, HC (3′)); 5.26 (dd, J=10.7, 1.5, 1H, HC(3′)); 4.9 (d, J=1.0, 1H, HC (4)); 4.74-4.72 (m, 2H, H₂C (1′)); 0.06 (s,9H, H₃CSi); ¹³C NMR (100 MHz) 164.01; 146.88; 131.79; 118.39; 104.24;65.75; -0.08. MS (EI) 200 (M+); 185; 157; 141; 115.

(b) General Synthesis of β,γ-Unsaturated Ketoesters

See, for example, (a) H. Sugimura, K. Yoshida, Bull. Chem. Soc. Jpn.1992, 65, 3209-3211. (b) D. A. Evans, J. S. Johnson, E. J. Olhava, J.Am. Chem. Soc. 2000, 122, 1635-1649. Borontrifluoride etherate (2.2equiv) was added over 5 min to a solution of aldehyde (1.0 equiv) inCH₂Cl₂ (0.5 M) at −78° C. The resulting solution was stirred for 30 min,then 2-(Trimethylsilanyloxy)-acrylic acid allyl ester (1.1 equiv) wasadded dropwise over 5 min. The solution was allowed to stir at −78° C.for 10 min, then warmed slowly to rt and stirred overnight. The mixturewas poured into sat. NaHCO₃ and extracted with EtOAc, the organic layerswere combined, dried (Na₂SO₄), filtered and the filtrate concentrated togive an oil which was dissolved in benzene (0.2 M) and silica gel (1 gper mmol) added). This mixture was heated to reflux for 2-4 h, cooled,filtered, the filter pad washed with EtOAc and the combined filtrateswere concentrated to give the crude unsaturated esters. Purification bycolumn chromatography then provided the pure unsaturated esters.

5-Methyl-2-oxo-hex-3-enoic acid allyl ester

¹H NMR (400 MHz) 7.14 (dd, J=15.6, 6.3, 1H, HC (4)); 6.60 (dd, J=15.6,1.5, 1H, HC (3)); 5.98 (ddt, J=17.6, 10.7; 5.9, 1H, HC (2′)); 5.41 (d,J=17.1, 1H, HC (3′)); 5.31 (d, J=10.7, 1H, HC (3)); 4.76 (dt, J=5.6,1.5, 2H, H₂C (1′)); 2.55 (m, 1H, HC (5)); 1.10 (d, J=6.8, 6H, H₃C (6));¹³C NMR (100 MHz) 183.22; 161.85; 160.67; 130.70; 122.25; 66.51; 31.76;20.90; MS (EI) 182 (M+); 109; 97; 87.

2-Oxo-4-phenyl-but-3-enoic acid allyl ester

¹H NMR (500 MHz) 7.87 (d J=16.1, 1H, HC (4)); 7.66 (d, J=8.3, 2H, HC(2′)); 7.45 (m, 3H, HAr); 7.36 (d, J=16.1, 1H, HC (3)); 6.02 (ddt,J=17.1, 10.8, 3.9, 1H, HC (2″)); 5.44 (dd, J=17.1, 1.5, 1H, HC (3″));5.35 (dd, J=10.7, 1.2, 1H, HC (3″)); 4.83 (d, J=5.9, 2H, H₂C (1″)); ¹³CNMR (100 MHz) 182.44; 161.72; 148.48; 133.86; 131.59; 130.78; 128.99;128.95; 120.43; 119.83; 66.74; MS (EI) 216 (M+); 131; 103.

4-(9H-Fluoren-2-yl)-2-oxo-but-3-enoic acid allyl ester

¹H NMR (500 MHz) 7.96 (d, J=16.1, 1H, HC (4)); 7.82 (m, 2H, HC (1′ and8′)); 7.67 (m, 1H, HAr); 7.59 (m, 1H, HAr); 7.43 (d, J=16.1, 1H, HC(3)); 7.44-7.35 (m, 3H, HAr); 6.05 (ddt, J=17.1, 10.3, 5.9, 1H, HC(2″)); 5.46 (d, J=17.1, 1H, HC (3″)); 5.36 (d, J=10.2, 1H, HC (3″));4.84 (dt, J=5.9, 1.5, 2H, H₂C (1″)); 3.95 (s, 2H, H₂C (9′)); ¹³C NMR(100 MHz) 182.17; 161.85; 148.92; 145.40; 143.98; 143.76; 140.44;132.36; 130.82; 128.63; 127.83; 127.00; 125.26; 125.10; 120.53; 120.23;119.79; 119.38; 66.79; 36.75; MS (EI) 304 (M+); 220; 219; 191; 189.

4-(3-Allyloxycarbonyl-3-oxo-propenyl)-benzoic acid methyl ester

¹H NMR (500 MHz) 8.08 (d, J=8.8, 2H, HC (2)); 7.87 (d, J=16.1, 1H, HC(1′)); 7.69 (d, J=8.3, 2H, HC (3)); 7.43 (d, J=16.1, 1H, HC (2′)); 6.07(ddt, J=17.1, 10.3, 6.3, 1H, HC (2″)); 5.44 (dd, J=17.1, 1.0, 1H, HC(3″)); 5.35 (dd, J=10.7, 1.3, 1H, HC (3″)); 4.82 (d, J=5.9, 2H, H₂C(1″)); 3.94 (s, 3H, H₃CO₂)); ¹³C NMR (100 MHz) 182.03; 166.00; 161.31;146.59; 137.86; 132.31; 130.61; 130.06; 128.66; 122.33; 119.97; 66.95;52.36; MS (EI) 274 (M+); 243; 189.

4-Benzo[1,3]dioxol-5-yl-2-oxo-but-3-enoic acid allyl ester

¹H NMR (500 MHz) 7.78)(d, 16.1, 1H, HC (4)); 7.14 (d, J=17.1, 1H, HC(3)); 6.85 (d, J=7.1, 1H, Har)); 7.14 (s, 1H, HC (6′)); 7.13 (d, J=7.2,1H, Har)); 6.04 (s, 2H, H₂CO₂); 6.01 (ddt, J=17.1, 10.7, 5.9, 1H, HC(2″)); 5.43 (dd, J=17.1, 1.0, 1H, HC (3″)); 5.33 (d, J=10.6, 1.0, 1H, HC(3″)); 4.81 (dt, J=5.9, 1.5, 2H, H₂C (1″)); ¹³C NMR (100 MHz) 182.01;161.82; 150.83; 148.43; 148.22; 130.80; 128.42; 126.49; 119.77; 118.39;108.68; 106.80; 101.79; 66.76; MS (EI) 260 (M+); 175.

2-Oxo-4-thiophen-3-yl-but-3-enoic acid allyl ester

¹H NMR (500 MHz) 7.86 (d, J=16.1, 1H, HC (4)); 7.69 (m, 1H, HAr); 7.40(m, 1H, HAr); 7.36 (s, 1H, HC (2′)); 7.16 (d, J=16.1, 1H, HC (3); 6.04(ddt, J=17.1, 10.7, 5.9, 1H, HC (2″)); 5.44, (dd, J=17.1, 1.5, 1H, HC(3″)); 5.34 (dd, J=10.6, 1.4, 1H, HC (3″)); 4.81 (dt, J=6.3, 1.0, 2H,H₂C (1″)); ¹³C NMR (100 MHz) 182.38; 161.56; 131.23; 130.66; 129.59;127.64; 127.23; 125.07; 120.08; 119.62; 66.12; MS (EI) 222 (M+); 137;109.

4-(Benzofuran-3-yl)-2-oxo-but-3-enoic acid allyl ester

¹H NMR (500 MHz) 7.74 (d, J=15.6, 1H, HC (4)); 7.62 (d, J=7.3, 1H, HAr);7.51 (d, J=8.3, 1H, HAr); 7.47 (d, J=15.6, 1H, HC (3)); 7.42 (td, J=7.3,1.0, 1H, HAr); 7.28 (td, J=8.3, 1.0, 1H, HAr)); 7.14 (s, 1H, HC (2′));6.03 (ddt, J=17.1, 10.3, 5.9, 1H, HC (2′)); 5.45 (dd, J=17.1, 1.0, 1H,HC (3″)); 5.36 (dd, J=10.2, 1.0, 1H, HC (3″)); 4.83 (dt, J=5.9, 1.5, 2H,H₂C (1″)); ¹³C NMR (100 MHz) 181.69; 161.26; 155.85; 152.04; 133.75;130.73; 128.19; 127.49; 123.54; 122.07; 120.43; 119.87; 114.69; 111.55;66.90; MS (EI) 256 (M+); 228; 213; 185; 129.

4-(1-Acetyl-1H-indol-3-yl)-2-oxo-but-3-enoic acid allyl ester

¹H NMR (500 MHz) 8.49 (d, J=7.8, 1H, HAr)); 8.03 (d, J=16.1, 1H, HC(4)); 7.91 (d, J=6.8, 1H, HAr); 7.86 (s, 1H, HC (2′)); 7.53 (d, J=16.1,1H, HC (3)); 7.48-7.40 (m, 2H. HAr); 6.21 (ddt, J=17.1, 10.7, 5.9, 1H,HC (2″)); 5.46 (dd, J=17.1, 1.5, 1H, HC (3″)); 5.36 (dd, J=10.7, 1.5,1H, HC (3″)); 4.84 (dt, J=5.9, 1.5, 2H, HC (1″)); 2.71 (s, 3H, H₃CC(O));¹³C NMR (100 MHz) 181.88; 168.14; 161.75; 139.69; 136.63; 130.77;130.14; 127.27; 126.35; 124.75; 120.23; 120.13; 119.85; 118.40; 116.87;66.87; 24.01; MS (ED) 297 (M+); 212; 170.

2-Oxo-4-(4-oxo-4H-chromen-3-yl)-but-3-enoic acid allyl ester

¹H NMR (500 MHz) 8.29 (dd, J=7.8, 1.5, 1H, HAr); 8.23 (t, J=8.8, 2H,HAr); 7.78 (m, 1H); 7.57 (d, J=15.1, 1H); 7.30 (m, 2H); 6.02 (ddt,J=17.1, 10.3, 6.3, 1H, HC (2″)); 5.44 (dd, J=17.1, 1.0, 1H, HC (3″));5.34 (dd, J=10.7, 1.5, 1H, HC (3″)); 4.82 (dt, J=5.9, 1.4, 2H, H₂C(1″)); ¹³C NMR (100 MHz) 183.08; 175.40; 161.39; 159.31; 155.23; 139.13;130.72; 126.23; 126.06; 124.01; 123.84; 119.76; 118.99; 118.08; 66.76;MS (EI) 284 (M+); 256; 228; 199; 149.

4-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-2-oxo-but-3-enoicacid allyl ester

¹H NMR (500 MHz) 7.92 (d, J=15.1, 1H, HC (4)); 7.68 (d, J=15.1, 1H, HC(3)); 7.50 (t, J=7.3, 2H, HAr); 7.39 (t, J=6.3, 1H, HAr); 7.31 (d,J=8.3, 2H, HAr); 5.96 (ddt, J=17.6, 10.7, 2.9, 1H, HC (2″)); 5.38 (dd,J=17.1, 1.5, 1H, HC (3″)); 5.27 (dd, J=10.7, 1.5, 1H, HC (3″)); 4.76 (d,J=3.0, 2H, H₂C (1″)); 3.32 (s, 3H, H₃CN); 2.42 (s, 3H, H₃CC); ¹³C NMR(100 MHz) 182.87; 162.68; 162.09; 153.01; 137.21; 133.46; 130.97;129.36; 128.20; 125.90; 119.34; 116.98; 103.62; 66.36; 34.46; 10.83; MS(EI) 326 (M+); 241.

(c) Building Block Testing

General procedure for test cycloadditions. Vinyl ether resin (preparedas described below) (5 mg, ˜1.1 meq/g, ˜0.005 mmol, 1 equiv), theappropriate heterodiene (0.015 mmol, 3 equiv) and 5 mg of activatedpowdered 4 Å molecular sieves were placed in a dry 4 mL vial, cappedwith septa and placed under Ar. THF (80 uL) was added, followed by a0.05 M solution of the appropriate catalyst 1 solution (prepared asdescribed below). The resulting mixture was vortexed gently for 16-24 hthen filtered (powdered sieves pass through the filter, thus separatingthem from the resin beads) and washed with 4×1 mL×20 min THF, then 3×1mL×15 min CH₂Cl₂ and dried briefly. The resin was then transferred to anEppendorf tube and treated with cleavage cocktail as described above,after concentration the samples were analyzed by ¹H NMR, LCMS and CSPHPLC and/or CSP SFC.

(d) Vinyl Ether Testing:

(4R,6R)-4-Benzo[1,3]dioxol-5-yl-6-(4-hydroxy-butoxy)-5,6-dihydro-4H-pyran-2-carboxylicacid allyl ester

¹H NMR (600 MHz) 6.74-6.72 (m, 2H, HAr); 6.67 (dd, J=7.8, 1.5, 1H,HAr)); 6.14 (d, J=2.9, 1H, HC (3)); 5.96 (ddt, J=17.1, 10.7, 5.3, 1H, HC(8′)); 5.93 (d, J=1.5, 2H, H₂CO₂)); 5.36 (dd, J=17.1, 1.5, 1H, HC (9′));5.26 (dd, J=10.7, 1.5, 1H, HC (9′)); 5.14 (dd, J=7.3, 2.0, 1H, HC (6));4.71 (ABX, J=14.3, 5.3, 2H, H₂C (7′)); 4.01 (dt, J=9.8, 5.9, 1H, HC(1″)); 3.70-3.60 (m, 4H, HC (4), HC (1″), H₂C (4″)); 2.28 (ddd, J=13.2,6.8, 1.5, 1H, HC (5)); 1.93 (dt, J=13.6, 7.8, 1H, HC (5)); 1.72-1.59 (m,4H, H₂C (2″); H₂C (3″)); MS (ESI) 400 (M+Na+1); 399 (M+Na); 387; 287;261; HRMS (ESI) C₂₀H₂₄O₇—Na requires 399.1420; found 399.1432.

(4R,6R)-4-Benzo[1,3]dioxol-5-yl-6-[2-(2-hydroxy-ethoxy)-ethoxy]-5,6-dihydro-4H-pyran-2-carboxylicacid allyl ester

¹H NMR (600 MHz) 6.72-6.69 (m, 3H, HAr); 6.23 (d, J=4.3, 1H, HC (3));6.0-5.96 (m, 1H, HC (8′)); 5.93 (d, J=5.4, 2H, H₂CO₂); 5.35 (dd, J=17.1,1.0, 1H, HC (9′)); 5.26 (dd, J=10.3, 1.0, 1H, HC (9′)); 5.16 (d, J=2.4,1H, HC (6)); 4.72 (ABX, J=18.6, 5.9, 5.9, 2H, H₂C (7′)); 3.94 (ddd,J=14.1, 9.8, 6.8, 1H, HC (1″)); 3.61-3.50 (m, 7H, HCO); 2.19 (m, 1H, HC(5). Other low field signals obscured by plasticizer.

(4R,6R)-4-Benzo[1,3]dioxol-5-yl-6-(4-hydroxymethyl-benzyloxy)-5,6-dihydro-4H-pyran-2-carboxylicacid allyl ester

¹H NMR (600 MHz) 7.36-7.30 (m, 4H, HAr); 6.74-6.70 (m, 2H, HAr); 6.66(dd, J=7.8, 1.5, 1H, HC (Ar)); 6.15 (d, J=2.9, 1H, HC (3)); 6.00-5.93(m, 1H, HC (8′)); 5.93 (s, 2H, H₂CO₂)); 5.37 (dd, J=17.1, 1.5, 1H, HC(9′)); 5.27 (dd, J=10.7, 1.5, 1H, HC (9′)); 5.20 (dd, J=7.3, 2.0 , 1H,HC (6)); 4.96 (d, J=11.7, 1H, HC (1″)); 4.78-4.68 (m, 5H, H₂C (6″); HC(1″); H₂C (7′)); 3.62 (ddd, J=10.7, 8.3, 2.9, 1H, HC (4)); 2.29 (ddd,J=14.3, 6.8, 1.5, 1H, HC (5)); 1.99 (dt, J=14.2, 7.8, 1H, HC (5)); MS(ESI) 447 (M+Na); 407; 363; 285; HRMS (ESI) C₂₄H₂₄O₇—Na requires447.1420; found 447.1401.

(4R,5S,6R)-6-Ethoxy-5-(3-hydroxy-propyl)-4-phenyl-5,6-dihydro-4H-pyran-2-carboxylicacid allyl ester

¹H NMR (600 MHz) 7.32-7.20 (m, 5H, HAr); 6.29 (d, J=3.9, 1H, HC (3));5.96 (ddt, J=17.1, 10.6, 5.9, 1H, HC (6′)); 5.36 (dd, J=17.1, 1.5, 1H,HC (7′)); 5.26 (dd, J=10.7, 1.5, 1H, HC (7′)); 4.76-4.65 (m, 2H, H₂C(5′)); 3.96 (dq, J=7.3, 7.1, 1H, HC (4″)); 3.7-3.5 (m, 3H, HC (4″) andH₂C (3″)); 3.49 (t, J=6.3, 1H, HC (4)); 2.25-2.20 (m, 1H, HC (5);1.60-1.22 (m, 4H, H₂C (1″) and H₂C (2″)); 1.19 (t, J=7.1, 3H, H₃C (5″));MS (ESI) 369 (M+Na); 357; 299; 280; 217; HRMS (ESI) C₂₀H₂₆O₅—Na requires369.1678; found 369.1669.

(4R,5R,6R)-6-Ethoxy-5-(3-hydroxy-propyl)-4-phenyl-5,6-dihydro-4H-pyran-2-carboxylicacid allyl ester

¹H NMR (600 MHz) 7.31-7.20 (m, 5H, HAr); 6.16 (d, J=3.4, 1H, HC (3));5.95 (ddt, J=17.1, 10.7, 5.9, 1H, HC (6′)); 5.35 (dd, J=17.1, 1.5, 1H,HC (7′)); 5.25 (dd, J=10.7, 1.4, 1H, HC (7′)); 4.92 (d, J=5.9, 1H, HC(6)); 4.71 (ABX, J=17.6, 5.4, 2H, H₂C (5′)); 4.00 (dq, J=7.3, 6.8, 1H,HC (4″)); 3.60-3.52 (m, 3H, H₂C (3″) and HC (4″)); 3.34 (dd, J=6.8, 3.4,1H, HC (4)); 2.03 (quint, J=6.9, 1H, HC (5)); 1.62-1.56 (m, 2H, H₂C(1″)); 1.50-1.46 (m, 2H, H₂C (2″)); 1.20 (t, J=7.3, 3H, H₃C (5″)); MS(ESI) 369 (M+Na); 299; 280; 217. HRMS (ESI) C₂₀H₂₆O₅—Na requires369.1678; found 369.1577.

(4R,5S,6R)-4-Benzo[1,3]dioxol-5-yl-6-ethoxy-5-{2-[2-(2-hydroxy-ethoxy)ethoxy]-ethyl}-5,6-dihydro-4H-pyran-2-carboxylicacid allyl ester

¹H NMR (600 MHz) 6.75-6.65 (m, 3H, HAr); 6.24 (d, J=3.9, 1H, HC (3));5.99-5.92 (m, 1H, HC (8′)); 5.93 (d, J=1.5, 1H, HCO₂)); 5.91 (d, J=1.5,HCO₂)); 5.35 (dd, J=17.1, 1.5, 1H, HC (9′)); 5.25 (dd, J=10.7, 1.5, 1H,HC (9′)); 5.19 (d, J=2.4, 1H, HC (6)); 4.77-4.66 (m, 2H, H₂C (7′));3.93-3.90 (m, 1H, HC (7″)); 3.78-3.30 (m, 12H, H₂CO; HC (7″); HC (4));1.19 (t, J=6.9, 3H, H₃C (8″)); other signals obscured; MS (ESI) 487(M+Na); 419; 417; 261; HRMS (ESI) C₂₄H₃₂O₉—Na 487.1944; found 487.1943.

(4R,5R,6R)-4-Benzo[1,3]dioxol-5-yl-6-ethoxy-5-{2-[2-(2-hydroxy-ethoxy)ethoxy]-ethyl}-5,6-dihydro-4H-pyran-2-carboxylicacid allyl ester

¹H NMR (600 MHz) 6.75-6.66 (m, 3H, HAr); 6.11 (d, J=3.4, 1H, HC (3));5.97-5.92 (m, 1H, HC (8′)); 5.93 (d, J=2.4, 2H, H₂CO₂)); 5.35 (dd,J=17.6, 1.5, 1H, HC (9′)); 5.25 (d, J 10.3, 1H, HC (9′)); 4.92 (d,J=5.9, 1H, HC (6)); 4.70 (ABX, J=13.6, 5.9, 2H, H₂C (7′)); 3.96 (qd,J=7.3, 6.8, 1H, HC (7″)); 3.75-3.43 (m, 11H, HCO; HC (7′)); 3.33 (dd,J=6.3, 3.4, 1H, HC (4)); 2.07, quint, J=5.9, 1H, HC (5)); 1.72-1.63 (m,2H, H₂C (1″)); 1.19 (t, J=6.8, 3H, H₃C (8″)); MS (ESI) 487 (M+Na); 419;261; HRMS (ESI) C₂₄H₃₂O₉—Na requires 487.1944; found 487.1921.

(4R,6R)-4-Benzo[1,3]dioxol-5-yl-6-{2-[(2-hydroxy-ethyl)-(4-methoxybenzenesulfonyl)-amino]-ethoxy}-5,6-dihydro-4H-pyran-2-carboxylicacid allyl ester

¹H NMR (600 MHz) 7.76 (d, J=8.8, 2H, HAr); 6.97 (d, J=8.8, 2H, HAr);6.74 (d, J=7.8, 1H, HAr)); 6.68-6.64 (m, 2H, HAr)); 6.13 (d, J=2.9, 1H,HC (3)); 6.00-5.90 (m, 1H, HC (8′)); 5.93 (s, 2H, H₂CO₂)); 5.35 (d,J=17.1, 1.5, 1H, HC (9′)); 5.26 (dd, J=10.7, 1.5, 1H, HC (9′)); 5.10(dd, J=8.3, 2.0, 1H, HC (6)); 4.77-4.65 (m, 2H, H₂C (7′)); 4.14-4.10 (m,1H); 3.92-3.84 (m, 1H, 3.86 (s, 3H, H₃CO); 3.71-3.68 (m, 2H); 3.64-3.62(m, 1H, HC (4)); 3.48 (dt, J=15.1, 4.4, 1H); 3.36-3.22 (m, 2H); 3.08(dt, J=14.6, 4.9, 1H); 2.26 (dd, J=12.7, 6.8, 1H, HC (5)); 1.86 (dt,J=13.2, 8.3, 1H, HC (5)); MS (ESI) 584 (M+Na); 287; 276. HRMS (ESI)C₂₇H₃₁NO₁₀S—Na requires 584.1566; found 584.1572.

(e) Heterodiene Testing:

(4S,6R)-6-{2-[(2-Hydroxy-ethyl)-(4-methoxy-benzenesulfonyl)-amino]-ethoxy}-4-isopropyl-5,6-dihydro-4H-pyran-2-carboxylicacid allyl ester

¹H NMR (600 MHz) 7.78-7.77 (m, 2H, HAr); 7.00-6.96 (m, 2H, HAr); 6.04(d, J=2.0, 1H, HC (3)); 5.87 (ddd, J=17.1, 10.7, 1.5, 1H, HC (10″));5.35 (dd, J=17.1, 1.5, 1H, HC (11″)); 5.25 (dd, J=10.7, 1.5, 1H, HC(11″)); 4.97 (dd, J=9.3, 1.3, 1H, HC (6)); 4.72-4.64 (m, 2H, H₂C (9″));4.14-4.10 (m, 1H); 4.02-3.99 (m, 1H); 3.96-3.90 (m, 1H); 3.87 (s, 3H,H₃CO); 3.80-3.70 (m, 2H); 3.50-3.32 (m, 2H); 3.29-3.26 (m, 1H);3.18-3.16 (m, 1H); 2.32-2.26 (m, 1H, HC (5)); 1.98-1.96 (m, 1H, HC (5));1.74-1.65 (m, 1H, HC (1′)); 0.96 (d, J=7.1, 3H, H₃C (2′)); 0.94 (d,J=7.1, 3H, H₃C (1′)); MS (ESI 506 (M+Na); 484 (M+1); 276; 258; HRMS(ESI) C₂₃H₃₃NO₈S—Na requires 506.1825; found 506.1847.

(4R,6R)-6-{2-[(2-Hydroxy-ethyl)-(4-methoxy-benzenesulfonyl)-amino]-ethoxy}-4-phenyl-5,6-dihydro-4H-pyran-2-carboxylicacid allyl ester

¹H NMR (600 MHz) 7.76-7.70 (m, 2H, HAr); 7.31-7.19 (m, 5H, HAr);7.00-6.96 (m, 2H, HAr); 6.20 (d, J=2.9, 1H, HC (3)); 5.93 (ddd, J=17.1,10.7, 1.6, 1H, HC (10″)); 5.36 (dd, J=17.1, 1.5, 1H, HC (11″)); 5.26(dd, J=10.7, 1.5, 1H, HC (11″)); 5.14 (dd, J=7.8, 2.0, 1H, HC (6));4.77-4.68 (m, 2H, H₂C (9″)); 4.15-4.10 (m, 1H); 4.02-3.98 (m, 1H);3.91-3.84 (m, 1H); 3.87 (s, 3H, H₃CO); 3.74-3.70 (m, 1H); 3.65-3.60 (m,1H); 3.50-3.40 (m, 1H); 3.29-3.22 (m, 2H); 3.08-3.00 (m, 1H); 2.31 (m,1H, HC (5)); 1.93 (dt, J=14.0, 7.5, 1H, HC (5)); MS (ESI) 540 (M+Na);518 (M+1); 276; 258; HRMS (ESI) C₂₆H₃₁NO₈S—Na requires 540.1668; found540.1644.

(4R,6R)-4-(9H-Fluoren-2-yl)-6-{2-[(2-hydroxy-ethyl)-(4-methoxybenzenesulfonyl)-amino]-ethoxy}-5,6-dihydro-4H-pyran-2-carboxylicacid allyl ester

¹H NMR (600 MHz) 7.77-7.70 (m); 7.55-7.53 (m); 7.38-7.20 (m); 7.70-6.94(m); 6.24 (d, J=2.9, 1H, HC (3)); 5.96 (ddd, J=17.1, 10.7, 1.5, 1H, HC(10″)); 5.36 (dd, J=17.1, 1.5, 1H, HC (11″)); 5.26 (dd, J=10.7, 1.5, 1H,HC (11″)); 5.17 (dd, J=7.8, 2.0, 1H, HC (6)); 4.77-4.68 (m, 2H, H₂C(9″)); 4.16-4.11 (m, 1H); 3.95-3.90 (m, 1H); 3.88-3.82 (m, 1H); 3.83 (s,3H, H₃CO); 3.82-3.80 (m, 1H, HC (4)); 3.64-3.62 (m, 1H); 3.51-3.46 (m,1H); 3.30-3.22 (m, 2H); 3.06-3.00 (m, 1H); 2.36 (dd, J=12.7, 7.3, 1H, HC(5)); 1.98 (dt, J=13.6, 8.8, 1H, HC (5)); MS (ESI) 628 (M+Na); 606(M+1); 536; 478; 276; HRMS (ESI) C₃₃H₃₅NO₈S—Na requires 628.1998; found628.2008.

(4R,6R)-6-{2-[(2-Hydroxy-ethyl)-(4-methoxy-benzenesulfonyl)-amino]-ethoxy}-4-(4-methoxycarbonyl-phenyl)-5,6-dihydro-4H-pyran-2-carboxylicacid allyl ester

¹H NMR (600 MHz) 7.98 (d, J=8.8, 2H, HC (3′)); 7.76-7.72 (m, 2H, HAr);7.28 (d, J=8.8, 2H, HC (2′)); 6.99-6.95 (m, 2H, HAr)); 6.19 (d, J=2.9,1H, HC (3)); 5.86 (ddd, J=17.1, 10.7, 1.5, 1H. HC (10″)); 5.36 (dd,J=17.1, 1.4, 1H, HC (11″)); 5.26 (dd, J=10.7, 1.5, 1H, HC (11″)); 5.17(dd, J=7.8, 2.0, 1H, HC (6)); 4.77-4.66 (m, 2H, H₂C (9″)); 4.14-4.08 (m,1H); 3.93-3.85 (m, 2H); 3.91 (s, 3H, H₃CO₂C)); 3.86 (s, 3H, H₃CO);3.79-3.76 (m, 1H, HC (4)); 3.62-3.60 (m, 1H); 3.46-3.42 (dt, J=15.1,4.9, 1H); 3.26-3.19 (m, 2H); 3.01 (dt, J=10.2, 5.9, 1H); 2.36-2.30 (m,1H, HC (5)); 1.96-1.92 (m, 1H, HC (5)); MS (ESI) 598 (M+Na); 576 (M+1);276; HRMS (ESI) C₂₈H₃₃NO₁₀S—Na requires 598.1723; found 598.1702.

(4R,6R)-6-{2-[(2-Hydroxy-ethyl)-(4-methoxy-benzenesulfonyl)-amino]-ethoxy}-4-thiophen-3-yl-5,6-dihydro-4H-pyran-2-carboxylicacid allyl ester

¹H NMR (600 MHz) 7.77-7.74 (m, 2H, HAr); 7.74-7.72 (m, 1H, HAr); 7.29(dd, J=4.9, 2.9, 1H, HAr); 7.05-6.95 (m, 3H, HAr); 6.21 (d, J=3.4, 1H,HC (3)); 5.86 (ddd, J=17.1, 10.7, 1.4, 1H, HC (10″)); 5.36 (dd, J=17.1,1.5, 1H, HC (11″)); 5.26 (dd, J=10.7, 1.5, 1H, HC (11″)); 5.15 (dd,J=7.8, 2.0, 1H, HC (6)); 4.75-4.64 (m, 2H, HC (9″)); 4.12-4.08 (m, 1H);4.02-3.98 (m, 1H); 3.92-3.80 (m, 2H); 3.86 (s, 3H, H₃CO); 3.74-3.70 (m,1H); 3.68 (t, J=4.9, 1H); 3.50-3.40 (m, 1H); 3.32-3.22 (m, 1H);3.07-3.02 (m, 1H); 2.34-2.30 (m, 1H, HC (5)); 2.00-1.95 (m, 1H, HC (5));MS (ESI) 546 (M+Na); 524 (M+1); 302; 276; 258; 249; HRMS (ESI)C₂₄H₂₉NO₈S₂ requires 546.1232; found 546.1230.

(4R,6R)-4-Benzofuran-3-yl-6-{2-[(2-hydroxy-ethyl)-(4-methoxybenzenesulfonyl)-amino]-ethoxy}-5,6-dihydro-4H-pyran-2-carboxylicacid allyl ester

¹H NMR (600 MHz) 7.74-7.70 (m, 2H, HAr); 7.48 (d, J=6.8, 1H, HAr); 7.42(d, J=8.3, 1H, Hr); 7.26-7.18 (m, 2H, HAr); 7.00-6.97 (m, 2H, HAr); 6.47(s, 1H, HC (2′)); 6.34 (d, J=3.9, 1H, HC (3)); 5.97 (ddd, J=17.1, 10.7,1.5, 1H, HC (10″)); 5.38 (dd, J=17.1, 1.5, 1H, HC (11″)); 5.24 (dd,J=10.1, 1.4, 1H, HC (11″)); 5.19 (dd, J=6.3, 2.4, 1H, HC (6)); 4.78-4.68(m, 2H, H₂C (9″)); 4.05-4.00 (m, 1H); 3.90-3.80 (m, 1H); 3.86 (s, 3H,H₃CO); 3.74-3.70 (m, 1H); 3.50-3.40 (m, 2H); 3.29-3.24 (m, 1H);3.20-3.16 (m, 1H); 3.13-3.07 (m, 1H); 2.98-2.90 (m, 1H); 2.37-2.25 (m,2H, H₂C (5)); MS (ESI) 580 (M+Na); 558 (+1); 258; HRMS (ESI)C₂₉H₃₁NO₉S—Na requires 580.1617; found 580.1595.

(4R,6R)-4-(1-Acetyl-1H-indol-3-yl)-6-{2-[(2-hydroxy-ethyl)-(4-methoxybenzenesulfonyl)-amino]-ethoxy}-5,6-dihydro-4H-pyran-2-carboxylicacid allyl ester

¹H NMR (600 MHz) 7.77-7.75 (m, 2H, HAr); 7.51-7.49 (m, 1H); 7.35-7.26(m, 3H, HAr); 6.95-6.90 (m, 2H, HAr); 6.21 (d, J=2.8, 1H, HC (3)); 5.91(ddd, J=17.1, 10.7, 1.4, 1H, HC (10″)); 5.36 (dd, J=17.1, 1.5, 1H, HC(11″)); 5.25 (dd, J=10.7, 1.5, 1H, HC (11″)); 5.19 (dd, J=8.4, 1.3, 1H,HC (6)); 4.68-4.61 (m, 2H, H₂C (9″)); 4.05-4.00 (m, 1H); 3.99-3.82 (m,2H); 3.85 (s, 3H, H₃CO); 3.84-3.80 (m, 1H); 3.60-3.56 (m, 1H); 3.45-3.20(m, 3H); 3.01 (dt, J=14.1, 5.1, 1H): 2.58 (s, 3H, H₃CC(O))); 2.38-2.33(m, 1H, HC (5)); 2.06-2.00 (m, 1H, HC (5)); MS (ESI) 621 (M+Na); 599(M+1); 276; HRMS (ESI) C₃₀H₃₄N₂O₉S—Na requires 621.1883; found 621.1860.

(4R,6R)-6-{2-[(2-Hydroxy-ethyl)-(4-methoxy-benzenesulfonyl)-amino]-ethoxy}-4-(4-oxo-4H-chromen-3-yl)-5,6-dihydro-4H-pyran-2-carboxylicacid allyl ester

¹H NMR (600 MHz) 8.25 (d, J=7.8, 1.5, 1 HAr); 7.87 (s, 1H, HC (2′));7.72 (d, J=9.3, 2H, HC (6″)); 7.68-7.64 (m, 1H, HAr); 7.46-7.40 (m, 2H,HAr); 6.97 (d, J=9.3, 2H, HC (7″)); 6.19 (d, J=3.9, 1H, HC (3)); 6.03(ddd, J=17.1, 10.7, 1.5, 1H, HC (10″)); 5.37 (dd, J 17.1, 1.5, 1H. HC(11″)); 5.28 (dd, J=10.7, 1.4, 1H, HC (11″)); 5.20 (dd, J=4.9, 2.4, 1H,HC (6)); 4.78-4.69 (m, 2H, HC (9″)); 4.02-3.98 (m, 1H, HC (4));3.88-3.72 (m, 2H); 3.86 (s, 3H, H₃CO); 3.58 (t, J=4.8, 1H); 3.36 (dt,J=14.7, 4.9, 1H); 3.32-3.24 (m, 1H); 3.17 (dt, J=15.1, 4.9, 1H);3.10-3.06 (m, 1H); 2.38-2.34 (m, 1H, HC (5)); 2.00-1.94 (m, 1H, HC (5));MS (ESI) 608 (M+Na); 586 (M+1); 311; HRMS (ESI) C₂₉H₃₁NO₁₀S—Na requires608.1566; found 608.1572.

(4R,6R)-4-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-6-{2-[(2-hydroxy-ethyl)-(4-methoxy-benzenesulfonyl)-amino]-ethoxy}-5,6-dihydro-4H-pyran-2-carboxylicacid allyl ester

¹H NMR (600 MHz) 7.76 (m, 2H, HAr); 7.46-7.30 (m, 5H, HAr); 6.99-6.96(m, 2H, HAr); 6.11 (d, J=2.9, 1H, HC (3); 5.96 (ddd, J=17.1, 10.7, 1.5,1H, HC (10″)); 5.35 (dd, J=17.1, 1.5, 1H, HC (11″)); 5.26 (dd, J=10.7,1.5, 1H, HC (11″)); 5.11 (dd, J=8.3, 2.0, 1H, HC (6)); 4.77-4.65 (m, 2H,H₂C (9″)); 4.04-3.99 (m, 1H); 3.90-3.85 (m, 1H); 3.86 (s, 3H, H₃CO);3.72-3.70 (m, 2H); 3.44-3.40 (m, 2H); 3.33-3.20 (m, 3H); 3.06 (s, 3H,H₃CN); 2.30-2.22 (m, 1H, HC (5)); 2.22 (s, 3H, H₃CC); 2.02-1.96 (m, 1H,HC (5)); MS (ESI) 650 (M+Na); 628 (M+1); HRMS (ESI) C₃₁H₃₇N₃O₉S—Narequires 628.2329; found 628.2348.

(D) Amine Building Blocks:

[4-(2-Amino-phenylcarbamoyl)-benzyl]-carbamic acid tert-butyl ester

¹H NMR (500 MHz, dmso-d₆) 9.60 (s, 1H, ArNH); 7.91 (d, J=7.8, 2H, HC(3″)); 7.48 (t, J=6.4, 1H, CH₂NH); 7.33 (d, J=6.8, 2H, HC (2″)); 7.15(d, J=7.8, 1H, HC (6′)); 6.96 (t, J=7.8, 1H, HC (4′)); 6.76 (d, J=7.8,1H, HC (3′)); 6.58 (t, J=7.3, 1H, HC (5′)); 4.88 (s, 2H, NH₂); 4.17 (d,J=6.4, 2H, H₂Car); 1.39 (s, 9H, H₃C); ¹³C NMR (100 MHz, dmso-d₆) 165.17;155.85; 143.78; 143.16; 133.06; 127.83; 127.56; 126.95; 126.66; 126.48;123.37; 116.28; 116.15; 77.94; 43.19; 28.26; MS (ESI) 342 (M+1); 286.

(g) Encoded Split-Pool Library Synthesis

Loading. A potion of the silyl functionalized resin 2 (1.43 meq/g, 2.10g, 3.0 mmol) was divided into eight equal portions (262 mg, 0.375 mmol,1.0 equiv), placed under argon in PD-10 tubes and suspended in 3 mL of3% (vol/vol) TMSCl/CH₂Cl₂ (in PD-10 columns). The swollen beads wereallowed to stand for 30 min, then filtered (under argon) and washed with4×3 mL×2 min CH₂Cl₂. Then, a solution of TfOH (3% in CH₂Cl₂, 6.6 mL,2.25 mmol, 6.0 equiv) was added and the resin was allowed to stand (withoccasional gentle mixing) for 25 min. The resin was filtered (underargon) and washed with 4×3 mL×2 min CH₂Cl₂. After the final wash anadditional 2 mL of CH₂Cl₂ was added to each pool, followed by2,6-lutidine (350 uL, 3.0 mmol, 8.0 equiv) and the resin was allowed tostand (with occasional gentle mixing) for 15 minutes. At this point eachof the vinyl ethers BB1A-H were added (0.75 mmol, 2.0 equiv) assolutions in 1 mL of CH₂Cl₂. The tubes were then allowed to stand for2.5 h (with occasional gentle mixing) then the resin was filtered andwashed with 4×3 mL×2 min CH₂Cl₂, then 1×5 mL×15 min THF, then 1×5 mL×15min CH₂Cl₂ then the resin pools were dried, first by simple suction for10 min, then under vacuum for 2 h and taken to the next step.

First Encoding Step. Each of the eight resin pools from above (0.375mmol, 1 equiv) was placed in a dry 8 mL vial capped with a septum. Toeach vial was added the appropriate diazoketone tags (see Table 3 below)(0.0672 mmol total tag for each reaction), followed by CH₂Cl₂ (4.0 mL,16.8 mM total tag concentration) was added to each vial and the vialswere gently shaken on a vortexer for 1 h. Then, a solution ofRh₂(O₂CPh₃)₄ (4 mL, 2.5 mg/mL) in CH₂Cl₂ was added to each vial and theresulting mixture vortexed for an additional 4 h, then the resin wasfiltered and washed with 2×5 mL×15 min CH₂Cl₂, then 1×5 mL×5 min THF,then 1×5 mL×8 h THF, then 2×5 mL×0 min THF, then 3×5 mL×15 min CH₂Cl₂.At this point the resin was pooled and rotated/washed in 1×15 mL×30 minTHF then 3×15 mL×30 min CH₂Cl₂, and dried as above to give 2.12 g oftagged vinyl ether. TABLE 3 Binary tagging scheme for first taggingstep. BB1- T2B (C4Cl3) T4B (C6Cl3) T1A (C3Cl5) T2A (C4Cl5) A 29.7 mg — —— B — 31.8 mg — — C — — 33.5 mg — D — — — 34.5 mg E 14.9 mg 15.9 mg — —F 14.9 mg — 16.7 mg — G 14.9 mg — — 17.2 mg H — 15.9 mg 16.7 mg —

Cycloaddition. The resin from above was divided into 20 equal portions(106 mg each, 0.15 mmol (theory), 1.0 equiv) and placed in dry 4 mLvials containing diene building blocks BB2A-J (2 vials per buildingblock, 0.45 mmol, 3.0 equiv) and 10 mg of activated powdered 4 Amolecular sieves. The vials were capped with septa and placed underargon. THF (0.8 mL) was added to each vial, followed by a solution ofappropriate catalyst solution (0.8 mL). (Catalyst solutions wereprepared by mixing 1 equiv of each2,2′-Isopropylidenebis[(4S)-4-t-butyl-2-oxazoline] ligand (141 mg) andCu(OTf)₂ (173 mg) with 4 Å molecular sieves (50 mg) in THF (12.8 mL) andstirred at rt temp to give a deep green mixture. An identical procedurewas used for the (R)-enantiomer of catalyst). The resulting mixtureswere vortexed gently for 20 h then filtered (powdered sieves passthrough the filter, thus separating them from the resin beads) andwashed with 4×5 mL×30 min THF, then 3×5 mL×15 min CH₂Cl₂ and dried asabove to give 20 pools of partially encoded cycloadducts.

Second Encoding Step. Each of the 20 resin pools (0.15 mmol (theory), 1equiv) was placed in a dry 8 mL vial capped with a septum. To each poolwas added the appropriate combination of tags (0.027 mmol total tag, seetable 4 below) followed by 1.6 mL of CH₂Cl₂ and the mixture vortexedgently for 1 h. Then, a solution of Rh₂(O₂CPh₃)₄ (1.6 mL, 2.5 mg/mL) inCH₂Cl₂ was added to each vial and the resulting mixture vortexed for anadditional 14 h, then the resin was filtered and washed with 2×5 mL×15min CH₂Cl₂, then 2×5 mL×15 min THF, then 1×5 mL×6 h THF, then 2×5 mL×15min THF, then 3×5 mL×15 min CH₂Cl₂. At this point the resin poolsderived from the (R) were combined (likewise the pools from the (S)catalyst were combined) and the two pseudo-enantiomeric pools wereindependently mixed /washed with 2×15 mL×30 min THF and 3×15 mL×15 minCH₂Cl₂, then filtered and dried to give two pseudo-enantiomeric pools ofresin, each containing roughly 1.43 g of fully encoded, resin boundcycloadducts. A portion of each pool ( 1/27) by weight was set aside atthis point to provide samples of the initial cycloadducts in the finallibrary collection. TABLE 4 Binary tagging scheme for second taggingstep. BB2- catalyst T3A (C5Cl5) T4A (C6Cl5) T5A (C7Cl5) T6A (C8Cl5) T7A(C9Cl5) A (S)-1 14.2 mg  — — — — B (S)-1 — 14.5 mg  — — — C (S)-1 — —14.9 mg  — — D (S)-1 — — — 15.3 mg  — E (S)-1 — — — — 15.7 mg  F (S)-17.1 mg 7.3 mg — — — G (S)-1 7.1 mg — 7.5 mg — — H (S)-1 7.1 mg — — 7.6mg — I (S)-1 7.1 mg — — — 7.9 mg J (S)-1 — 7.3 mg 7.5 mg — — A (R)-1 —7.3 mg — 7.6 mg — B (R)-1 — 7.3 mg — — 7.9 mg C (R)-1 — — 7.5 mg 7.6 mg— D (R)-1 — — 7.5 mg — 7.9 mg E (R)-1 — — — 7.6 mh 7.9 mg F (R)-1 4.7 mg4.8 mg 5.0 mg — — G (R)-1 4.7 mg 4.8 mg — 5.1 mg — H (R)-1 4.7 mg 4.8 mg— — 5.2 mg I (R)-1 4.7 mg — 5.0 mg 5.1 mg — J (R)-1 4.7 mg — 5.0 mg —5.2 mg

Deallylation. Each of the two resin pools from above (1.5 mmol (theory)1.0 equiv) was treated identically.Tetrakis(triphenylphosphine)palladium (1.73 g, 1.5 mmol, 1.0 equiv) wasdissolved in 26 mL of THF. The dry resin was then added to this solutionfollowed by thiosalicylic acid (1.62 g, 10.5 mmol, 7 equiv) and themixture was vortexed gently for 12 h, then the resin pools were filteredand each washed separately with 4×15 mL×1 h THF, then 2×15 mL×15 minDMF, then 1×15 mL×15 min THF, then 1×15 mL×15 min DMF, then 4×15 mL×15min CH₂Cl₂ then dried to give two pools of resin each weighing roughly1.34 g. A portion of each pool ( 1/26 by weight) was set aside at thispoint to provide samples of the cycloadduct carboxylic acids in thefinal library collection.

Amide Formation. Each of the two pools from above were split into 25equal portions (0.056 mmol (theory), 1 equiv), then each set of 25 wastreated identically. To each portion of resin in a 4 mL vial was added astock solution of PyBop (193 mg/mL in CH₂Cl₂, 1.5 mL, 0.55 mmol, 10equiv) followed by stock solutions of the amine building blocks (1.1 Min DMF, 500 uL, 0.56 mmol, 10 equiv). Then, diisopropylethylamine (100uL, 0.56 mmol, 10 equiv) was added to each vial and the resultingmixtures were vortexed for 12 h. Each reaction mixture was then filteredand washed with 2×1 mL×30 min CH₂Cl₂, then 2×1 mL×30 min DMF, then 3×1mL×30 min THF, then 3×1 mL×30 min CH₂Cl₂ then dried as above to give 50spatially segregated pools of dihydropyrancarboxamides plus the fourpools from above for both (R) and (S)-derived esters and acids. Thesesamples were kept separate to allow for “spatial coding” of the aminebuilding block, in addition to the chemical encoding of the first andsecond building blocks.

Example 2 Solid Support Synthesis of Inventive Compounds DecodingMethodology

Discussion of Methodology

As described above and in Scheme 4, an encoded split-pool library of4320 dihydropyrancarboxamides (12) was synthesized featuring an R- orS-bis(oxazoline)copper (II) triflate-catalyzed heterocycloadditionreaction as a diversity-generating step. The three sets of BBs used inthe library synthesis are shown in FIG. 3. As discussed above, thelibrary synthesis was based on three chemical steps: (1) loading ofeight vinyl ethers onto the PS beads (4), (2) enantioselectivecycloaddition with 10 β,c-unsaturated ketoesters, followed by allylester deprotection, and (3) PyBOP(benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate)-mediated coupling (See J. Coste, D. Lenguyen, B.Castro, PyBOP: a new peptide coupling reagent devoid of toxic byproduct,Tetrahedron Lett. 1990, 31:205-208) to 25 different amines to yieldsupport-bound dihydropyrancarboxamides (11).

Reaction step and BB encoding were carried out twice in the librarysynthesis: first, after loading the eight vinyl ethers, and second,after the cycloaddition reaction with ten heterodienes. The tags andbinary codes used for each BB are shown in Tables 5 and 6. In thecycloaddition reaction, one set of beads (10 portions) was treated withthe (S)—Cu (II) catalyst, and the other ten portions were treated withthe (R)-catalyst (Scheme 4). From this step onwards, these two groups ofenantiomers were kept separate even though they were encoded for eachenantiomer of the catalyst used. The subsequent reactions were carriedout in parallel so that spatial decoding could be performed had thechemical encoding failed. The 25 final amide pools were kept separate toreduce the number of chemical encoding steps. This library synthesisresulted in 54 (27×2 enantiomers) separate portions of solid supports(11) containing, theoretically, three copies of 4320 stereochemicallyand structurally distinct compounds (12). Finally, as our macrobeadhandling ‘best practices’ were observed throughout the librarysynthesis, the majority of the library supports remained intact (>90%).In order to test the integrity of our optimized libraryencoding/decoding protocol, 108 macrobeads from the library(theoretically 2.5% of the total library compounds) were arrayed intotubes and treated with HF/py, followed by TMSOMe to release thecompounds (12) from the beads. The residue isolated from each bead wasdissolved in CH₃CN and transferred to individual glass autosamplerinserts to provide arrayed stock solutions of small molecules. Afraction of each of these stock solutions was subjected to LC/MSanalysis, and the corresponding macrobeads were submitted to ouroptimized decoding protocol to compare the two results (FIG. 7). TABLE 5Binary codes for encoding BB1 of library 12 Entry Tag C4Cl3 Tag C6Cl3Tag C3Cl5 Tag C4Cl5 BB1-A 1 0 0 0 BB1-B 0 1 0 0 BB1-C 0 0 1 0 BB1-D 0 00 1 BB1-E 1 1 0 0 BB1-F 1 0 1 0 BB1-G 1 0 0 1 BB1-H 0 1 1 0

TABLE 6 Binary codes for encoding BB2 of library 12 inheterocycloadditions catalyzed by either R- or S-bis(oxazoline)copper(II) tri£ate Entry Catalyst Tag C5Cl5 Tag C6Cl5 Tag C7Cl5 Tag C8Cl5 TagC9Cl5 BB2-A S 1 0 0 0 0 BB2-B 0 1 0 0 0 BB2-C 0 0 1 0 0 BB2-D 0 0 0 1 0BB2-E 0 0 0 0 1 BB2-F 1 1 0 0 0 BB2-G 1 0 1 0 0 BB2-H 1 0 0 1 0 BB2-I 10 0 0 1 BB2-J 0 1 1 0 0 BB2-A R 0 1 0 1 0 BB2-B 0 1 0 0 1 BB2-C 0 0 1 10 BB2-D 0 0 1 0 1 BB2-E 0 0 0 1 1 BB2-F 1 1 1 0 0 BB2-G 1 1 0 1 0 BB2-H1 1 0 0 1 BB2-I 1 0 1 1 0 BB2-J 1 0 1 0 1

Decoding consisted of deriving the identities of BBs 1 and 2 by GC taganalysis, adding their combined molecular weight to that of the aminecorresponding to the pool of supports from which the macrobead wastaken, and comparing this composite mass to the mass observedexperimentally by APCI/MS. The structural data obtained via GC decodingwere in complete agreement with the MS data obtained from the compounds'stock solutions (FIGS. 6A-I) for 107 of the 108 samples. Seventy of the108 macrobeads (65%) yielded GC traces that decoded for a compound witha molecular ion identical to that expected based on the MS data.Twenty-five macrobeads (23%) showed GC traces that decoded for acompound whose molecular ion corresponded to a fragment of the proposedstructure. Direct stock solution decoding, using the optimized decodingprotocol on a fraction (˜5%) of the stock solutions generated fromindividual macrobeads, was carried out successfully to identify thestructures of the 12 remaining samples (See H. E. Blackwell, L. Pérez,S. L. Schreiber, “Decoding products of diversity pathways from stocksolutions derived from single polymeric macrobeads”, Angew. Chem. Int.Ed. 2001, 40:3421-3425). Structures of 25 representative compounds fromthe 108 beads decoded of library 12 are shown below (numbers in boldrefer to bead number). All structures show agreement between their GCand MS decoding data. Representative GC, LC, and MS traces for a singlemacrobead are shown in FIGS. 9A-B. In addition, LC and MS traces for 25inventive compounds (quality control compounds) are depicted in FIGS. 16(16A-16D) and 17 (17A-17C).

The successful synthesis and partial decoding of library 12 validate notonly our binary encoding/decoding protocol, but also the entiresynthesis platform as a reliable procedure for the generation of encodedsplit-pool libraries. The use of stock solution decoding further enablesthis platform as it simplifies the elucidation of structures of ‘hits’from assays and lends itself to future automation.

The successful synthesis of an encoded split-pool library (12) usingthis platform validates the approach. The synthesis platform usescommercially available reagents and straightforward syntheticprocedures; therefore, we believe it could be readily established inother laboratories. This work lays the foundation for the second phaseof platform development, where the members of libraries are distributedon a per bead basis into multiwell assay plates, submitted to automatedcleavage, and resuspended to generate plates of pure, arrayed stocksolutions, as described in Example 3 (See also P. A. Clemons, A. N.Koehler, B. K. Wagner, T. G. Sprigings, D. R. Spring, R. W. King, S. L.Schreiber, M. A. Foley, “A one-bead, onestock solution approach tochemical genetics, part 2”, Chem. Biol. 2001 , 8:1183-1195). Theindividual stock solutions originating from single macrobeads have beenfound to be sufficient for hundreds of phenotypic assays (forwardchemical genetics) and thousands of protein-binding assays (reversechemical genetics) before a need for re-synthesis (FIG. 10).

Experimental Section

I. General Synthetic Methods

General Methods. Reagents were obtained from Aldrich Chemical Co.,Acros, Novabiochem, or J. T. Baker and used without furtherpurification. Reaction solvents (THF, Et₂O, DMF, toluene, and CH₂Cl₂)were obtained from J. T. Baker (HPLC grade) and purified by passagethrough two solvent columns prior to use. The CH₂Cl₂ and toluenepurification systems were composed of one activated alumina (A-2) columnand one supported copper redox catalyst (Q-5 reactant) column. The THFand Et₂O purification systems were composed of two activated alumina(A-2) columns, and the DMF purification system was composed of twoactivated molecular sieve columns. See: Pangborn, A. B.; Giardello, M.A.; Grubbs, R. H.; Rosen, R. K.; Timmers, F. J. Organometallics 1996,15, 1518-1520. Diisopropylethylamine (DIPEA) and 2,6-lutidene weredistilled from calcium hydride; MeOH was distilled from magnesiummethoxide. Brominated polystyrene (Br—PS, 2 mequiv/g) was obtained fromPolymer Labs (Product #:1462-9999, $18/g). Solution phase reactions wereperformed in oven- or flame-dried glassware under positive N₂ pressure.

Solid Phase Reactions. Small-scale solid phase reactions (5-10 mg resin)were performed in 500 μL polypropylene Eppendorf tubes with mixingprovided by a Vortex Genie-2 vortexer fitted with a 60 microtube insert.Medium-scale solid phase reactions (20-500 mg resin) were performed in 2mL fritted polypropylene Bio-Spin® chromatography columns (Bio-Rad) or10 mL fritted polypropylene PD-10 columns (Pharmacia Biotech) with 360°rotation on a Barnstead-Thermolyne Labquake™ Shaker. Large-scale solidphase reactions (>500 mg resin) were performed in silanized 50 or 100 mLfritted glass tubes equipped for vacuum filtration and N₂ bubbling. Thetubes were silanized by treatment with 20% dichlorodimethylsilane/CH₂Cl₂for 15 min, MeOH for 15 min, followed by oven heating at 120° C. for atleast 2 h.

After small-scale reactions, resin samples were transferred to 2 mLBioSpin® columns. Resin samples in polypropylene columns were washed ona Vac-Man® Laboratory Vacuum Manifold (Promega) fitted with nylon 3-waystopcocks (Bio-Rad). Resin samples in glass tubes were washed in thetubes with alternating periods of N₂ bubbling and vacuum draining. Thefollowing standard wash procedure was used: 3×THF, 3×DMF, 3×THF,3×CH₂Cl₂.

Resin samples were then transferred via spatula to 500 μL Ependorf tubesand suspended in Ar-degassed HPLC grade THF followed by pyridine andhydrogen fluoride-pyridine (Aldrich, HF (70%)/pyridine (30%)) in a ratioof 90:5:5. Samples were then sealed with parafilm and gently agitated ona vortexer for 30 min. Methoxy-trimethylsilane (TMSOMe) was added andthe samples were sealed with Parafilm and placed on a vortexer for anadditional 30 min. The supernatant fluid was removed, transferred toanother Eppendorf tube, and concentrated in vacuo.

Purification and Analysis. Flash chromatography was performed on E.Merck 60 230-400 mesh silica gel. TLC was performed on 0.25 mm E. Mercksilica gel 60 F₂₅₄ plates and visualized by UV (254 nm) and ceriumammonium molybdate. HPLC was performed on a Nest Group (Southborough,Mass.) Hypersil C18 100 Å 3 μM, 4.6 mm×6 cm column using a flow rate of3 mL/min and a 4 min gradient of 0-99.9% CH₃CN in H₂O/0.1% TFA, constant0.1% MeOH with diode array UV detection. IR spectra were recorded on aNicolet 5PC FT-IR Spectrometer or a Bruker Vector 22 Spectrometer withpeaks reported in cm⁻¹. NMR spectra were recorded on Varian Inova 500MHz and 400 MHz instruments. Solid-phase NMR spectra were recorded on aVarian Inova 500 MHz equipped with a Nanoprobe (See (a) Fitch, W. L.;Detre, G.; Holmes, C. P.; Shoolery, J. N.; Keifer, P. A. J. Org. Chem.1994, 59, 7955-7956. (b) Keifer, P. A.; Baltusis, L.; Rice, D. M.;Tymiak, A. A.; Shoolery, J. N. J. Magn. Reson., Series A 1996 119,65-75). Chemical shifts are expressed in ppm relative to TMS (0.00 ppm)or residual solvents. Peak assignments were made based on homonucleardecoupling and/or two-dimensional DQF-COSY, TOCSY, and/or NOESYexperiments. Mass spectra were obtained on JEOL AX-505H or SX-102A massspectrometers by electron impact ionization (EI), chemical ionization(CI) with ammonia (NH₃), or fast atom bombardment ionization (FAB) withglycerol or 3-nitrobenzyl alcohol/sodium iodide (NBA/NaI) matrices.LC/MS data was obtained on a Micromass Platform LCZ mass spectrometer inatmospheric pressure chemical ionization (APCI) mode attached to aWaters 2690 HPLC system. LC/MS chromatography was performed on a WatersSymmetry C18 3.5 μM, 2.1 mm×50 mm column using a flow rate of 0.4 mL/minand a 10 min gradient of 15-100% CH₃CN in H₂O, constant 0.1% formic acidwith 200-450 nm detection on Waters 996 photodiode array detector.GC/ECD data was obtained on a Hewlett Packard 6890 Gas Chromatographfitted with a 7683 series injector and autosampler, split-splitlessinlet, μ-ECD detector, and a J&W DB1 15 m×0.25 mm×0.25 μm column.(Gradient start temperature: 110° C.; hold 1 min, ramp 45° C./min to250° C., hold 2 min, ramp 15° C./min to 325° C., hold 2 min. Flow rate:constant flow, 1 mL/min. Inlet is purged at 1 min with flow rate 60mL/min, reduced to 20 mL/min at 2 min).

II. Allyl Silane Linker Synthesis

Diisopropyl(4-methoxyphenyl)silane. A solution of p-bromoanisole (28.6mL, 228 mmol, 1.0 equiv.) in THF (550 mL) was chilled to −78° C.(CO₂(s), acetone) and treated with n-BuLi (91.2 mL, 228 mmol, 2.5 M inhexanes, 1 equiv.) via cannula over a 5 min period. After 5 min a whiteprecipitate began to form. The mixture had stirred for 30 min at −78° C.when diisopropylchlorosilane (34.6 g, 228 mmol, 1.0 equiv.) was slowlyadded via syringe. After 1 h the ice bath was removed, and the solutionwas allowed to come to 23° C. with continued stirring overnight. Themixture was treated with saturated NH₄Cl (50 mL) and extracted withether (3×500 mL). The combined organic extracts were washed with brine,dried over MgSO₄, filtered and concentrated in vacuo to yield a lightyellow oil. Silica gel chromatography (gradient: 3-5% EtOAc/hexanes)yielded (47.7 g, 94%) of a colorless oil. This material could also bepurified by distillation [BP=76-85° C. @ 275 mTorr (40 g, 63%)]. TLCR_(f)=0.61 (1:9 EtOAc/hexanes). IR (film): 2393, 1853, 1710, 1691, 1658,1584, 1482, 1346. ¹H NMR (500 MHz, CDCl₃): δ7.48 (d, 2H, J=8.10), 6.95(d, 2H, J=8.10), 3.97 (s, 1H, Si—H), 3.85 (s, 3H), 1.39 (q, 2H, J=3),1.10 (d, 6H, J=6.5), 1.03 (d, 6H, J=7.5). ¹³C NMR (126 MHz, CDCl₃): δ137.13, 113.73, 113.62, 55.18, 18.95, 18.72, 11.08. Elemental analysis,Calcd.: C, 70.21; H, 9.97; Si, 12.63. Found: C, 70.43; H, 9.83; Si,12.39.

Chloro(4-methoxyphenyl)diisopropylsilane.Diisopropyl(4-methoxyphenyl)silane (47.7 g, 214 mmol, 1.0 equiv.), wastaken up in CH₂Cl₂ (700 mL). The solution was cooled to 0° C. andtrichloroisocyanuric acid (16.6 g, 71.3 mmol, 0.33 equiv.) was carefullyadded in three equal portions, making sure that each portion had atleast 7 min to react before the next was added. (Caution! Addingtrichloroisocyanuric acid too rapidly results in a rapid evolution ofgas and concomitant expulsion of the reaction vessel contents). Themixture was stirred at 0° C. for 40 min, followed by warming to 23° C.with stirring. The solids were filtered under an inert atmosphere, andthe filtrate was concentrated in vacuo to yield 54.8 g (98%) of a cloudyoil. The chlorosilane, which is unstable, was used immediately andwithout purification in the next step.

Allyl(4-methoxyphenyl)diisopropylsilane. To the crudechloro(4-methoxyphenyl)-diisopropylsilane (54.8 g, 214 mmol, 1.0 equiv.)was added THF (335 mL) via cannula under Ar. The solution was chilled to0° C. and treated with allylmagnesium chloride (128 mL, 256 mmol, 2.0 Min THF, 1.2 equiv.). After 3 h at 0° C., the solution was allowed towarm to 23° C. with stirring overnight (16 h). The mixture was treatedwith saturated NH₄Cl (50 mL), and the aqueous layer was extracted withether (3×500 mL). The combined organic extracts were washed with brine,dried over MgSO₄, filtered, and concentrated in vacuo. The crudematerial was purified by silica flash chromatography (3-5%EtOAc/hexanes) to yield 52.86 g (94%) of a slightly cloudy, clearviscous oil. This reagent distills at 80° C. at 500 mTorr as a colorlessoil. TLC R_(f)=0.40 (1:9 EtOAc/hexanes). IR (film): 2942, 2865, 1630,1595, 1504, 1463, 1277. ¹H NMR (500 MHz, CDCl₃): δ 7.32 (d, 2H, J=6.84),6.81 (d, 2H, J=6.84), 5.82 (q, 1H, J=8.5, 8.5), 4.88 (d, 1H, J=17.05),4.76 (d, 1H, J=9.77), 1.82 (d, 2H, J=7.32), 1.17 (q, 2H, J=7.3), 0.94(d, 6H, J=7.3), 0.90 (d, 6H, J=7.3). ¹³C NMR (126 MHz, CDCl₃): δ 160.51,136.48, 135.70, 125.78, 113.78, 113.62, 55.09, 19.34, 18.22, 18.17,17.68, 11.30. Elemental analysis, Calcd.: C, 73.22; H, 9.98; Si, 10.70.Found: C, 73.25; H, 9.97; Si, 10.77.

III. PS Resin Derivatization

Hydroboration of Allyl(4-methoxyphenyl)diisopropylsilane. Solid 9-BBNdimer (6.29 g, 53.0 mmol, 0.95 equiv.) was weighed out in a glove boxand sealed under an Ar atmosphere. Freshly distilled THF (365 mL) andallyl(4-methoxyphenyl)diisopropylsilane (14.64 g, 55.8 mmol, 1.0 equiv.)were added via syringe, and the mixture was allowed to stir for 3 h at23° C. The overall concentration of theallyl(4-methoxyphenyl)diisopropylsilane in THF was 0.16 M, which was theappropriate concentration for the subsequent Suzuki coupling. The yieldof this reaction was assumed to be quantitative.

Suzuki Coupling. To the alkyl-borane containing THF solution above (53.0mmol in 365 mL of THF, 1.74 equiv.) was added the solid Br—PS (15.25 g,2 mequiv/g 30.5 mmol of Br, 1.0 equiv.) Care was taken to maintain an Arblanket over the solution. Br—PS was allowed to swell for 45 min, andthen treated with tetrakis(triphenylphosphine)palladium(0) (880 mg, 0.76mmol, 0.025 equiv.) followed by aqueous NaOH solution (61 mmol, 30.5 mLof a 2M NaOH solution, 2.0 equiv.). The reaction was then heated toreflux with gentle stirring for 24 h. Pd(0) (880 mg, 0.76 mmol, 0.025equiv.) was added, and the reaction was heated to reflux for another 12h. The biphasic reaction mixture turned slightly green from its initialyellow color. The mixture was filtered, and the beads were washedrepeatedly (see below). While it was unnecessary to agitate the beadsduring the wash cycle, it was critical to allow the beads sufficienttime to absorb the washing solvent. Wash procedure: THF (2×100 mL×45min), 3:1 THF/1 M NaCN (1×100 mL×1 h or until all dark color is gone),3:1 THF/H₂O (2×100 mL×45 min), 3:1 THF/IPA (2×100 mL×45 min), THF (2×100mL×45 min), CH₂Cl₂ (2×100 mL×45 min). The beads were air-driedovernight, then placed on a lyophilizer for 24 h, producing an almostcolorless, opaque resin. ¹H NMR (500 MHz, nanoprobe, CD₂Cl₂ gel phase):δ 7.34 (m, 4H), 6.82 (m, 4H), 3.69 (s, 3H), 1.76 (m, 2H), 1.22 (m, 2H),1.16 (m, 2H), 0.97 (m, 2H), 0.91 (m, 12H) [For a discussion of theeffect of resin linker length on gel-phase NMR spectral line widths,see: Keifer, P. A. J. Org. Chem. 1996, 61, 1558-1559]. Elementalanalysis. Found C, 83.54; H, 8.28; Si, 4.35; Br, <0.02; Cl, 0.247.

Determination of Bead Loading by Elemental Analysis. 2.0 mmolp-bromopolystyrene beads, quantitatively loaded with the silicon linkerabove, contain 41 mg Si/g resin or 4.1% Si. Assuming quantitativeloading, the mass of 1 g resin would increase to 1.37 g; therefore, thelinker loading is calculated as 1.45 mequiv/mol. Thus, the resin loadingis estimated from two elemental analyses parameters, % Si and % Br. The% Br<0.02 by weight indicates qualitative disappearance of Br (note thathalogens can be confused by elemental analysis, hence it is necessary toperform separate Br and Cl analysis), while percent Si indicates theloading level. Percent Si typically ranges from 3.79 to 4.05%. Theprocedure used to calculate percent Si can overestimate the actualamount of Si by 0.2-0.3% as these numbers are calculated by weighing ashresultant from sample digestion with acid and residue combustion, whichleaves some elements unresolved from Si. 4.35% Si is equivalent to 43.5mg Si/g resin, or 1.54 mequiv Si/g. The actual loading used insubsequent calculations was 1.45 mequiv/g, the theoretical maximum.There were 9,350 beads/g of 500-600 copolymerized p-bromopolystyrenebeads with 2.0 mmol Br/g loading level. We assumed quantitativeconversion, justified by disappearance of bromine and appearance ofappropriate amount of silicon. Thus, the number of polystyrene beads inone gram of resin was then scaled with a 37% mass increase, or about6,800 beads/g.

IV. Bead Stability Studies (FIG. 4)

While the use of sequences of tandem organic reactions can efficientlygenerate complex molecules in diversity-oriented syntheses [S. L.Schreiber, Target-oriented and diversity-oriented organic synthesis indrug discovery, Science 287 (2000) 1964-1969], we have observed thatsuccessive organic transformations, coupled with rigorous bead washingbetween reactions, can damage the PS macrobeads. The exemplarytechnology platform used for the present study (FIG. 10), however,implies that we isolate one physically intact bead per well prior tocompound cleavage for several reasons. First, fragments of beads yieldweaker compound stock solutions after bead arraying, cleavage, andresuspension. Second, the possibility of isolating more than onefragment per well allows for stock solution contamination and theconcomitant incorrect decoding of that well. To avoid these problems, wehave developed a set of standard practices for bead handling duringlibrary synthesis and encoding that dramatically minimize thepossibility of bead breakage.

In general, we have found that the less we handle the solid supportsphysically, either by submission to chemical reactions, washing, ordrying, the less bead breakage we observe. This reinforces theimportance of an effective planning algorithm for diversity-orientedsyntheses. Short reaction sequences yielding complex and diversecompounds not only ensure that positives can be re-synthesized readily,but also promote the integrity of the beads. In order to quantify beadintegrity, we used population size distribution measurements (obtainedby light obscuration) to monitor the shift of the average particle sizein a sample of beads (data not shown). We first observed that the PSmacrobeads were fragile when swollen in organic solvents. Since the useof solvents and drying are required in library synthesis, we assessedseveral solvent, drying, and agitation conditions. Even though certainchemical transformations appear to cause more bead breakage than others,we did not include different chemical reactions as experimentalvariables in our studies because we did not want to limit the types ofchemistry utilized in library synthesis.

As evidence that even the most simple and gentle handling inducesdamage, supports swollen in dichloro-methane (CH₂Cl₂) and drained seventimes, followed by overnight air drying resulted in a shift to a smalleraverage size distribution. As an example of extreme damage, beads weresubjected to swelling in tetrahydrofuran (THF) (45 min), followed bytreatment with methanol (MeOH) (45-min) and 360° rotation. The beadswere then rapidly dried via lyophilization, and the whole process wasrepeated seven times. These supports show even more extensive damage anda greater degree of bead fragmentation. The ‘best practices’ weextrapolated from these experiments include light agitation from awrist-action shaker, followed by blowing N₂ over the resin (30 min), andfinal drying under high vacuum conditions from any organic solvent.While a shift in average size still exists, these conditions minimizefragmentation and are suitable for library syntheses, as judged by ourability to array one intact bead per well after library synthesis (seeFIG. 4) [See S. M. Sternson, J. B. Louca, J. C. Wong, S. L. Schreiber,Split-pool synthesis of 1,3-dioxanes leading to arrayed stock solutionsof single compounds sufficient for multiple phenotypic andprotein-binding assays, J. Am. Chem. Soc. 123 (2001) 1740-1747; and P.A. Clemons, A. N. Koehler, B. K. Wagner, T. G. Sprigings, D. R. Spring,R. W. King, S. L. Schreiber, M. A. Foley, A one-bead, one-stock solutionapproach to chemical genetics, part 2, Chem. Biol. 8 (2001) 1183-1195].

V. Library Encoding and Decoding Protocols

Representative Bead Encoding Procedure. Place 20 dry beads(approximately 3 mg resin) in a 700 μL Eppendorf tube. Prepare a fresh8.4 mM (in each tag) solution in dry CH₂Cl₂ in an oven-dried, Tefloncapped glass vial. (NOTE: The tag concentration can be cut by one-halfto one-fifth, and the tags will still be readable by GC (the late tagswill be weak). This might be necessary for large library syntheses wherea large quantity of tag is required, or if more than 4 tags are used ineach tagging step. Use the same volume of tag solution as describedbelow.) Add 50 μL of the tag solution to the Eppendorf tube. Set thetube to shake for 45 min at room temperature on a tabletop orbitalshaker. Prepare a 4.4 mg/mL solution of the catalyst, rhodiumtriphenylacetate (Rh₂(O₂CC(Ph)₃)₄), in dry CH₂Cl₂ under Ar in anoven-dried, Teflon capped glass vial. (NOTE: The catalyst concentrationcan be cut by one-half to one-fifth and the tags will still be readableby GC (the late tags will be weak). Use the same volume of catalystsolution as described below.) Add 50 μL of the catalyst solution to theresin and keep the Eppendorf in agitation for 16 h (overnight) at roomtemperature. Wash the resin in a 1 mL BioRad tube 2×15 min CH₂Cl₂, 16 h(overnight) THF, 2×15 min THF, and 2×15 min CH₂Cl₂. Dry the resin underhouse vacuum for ca. 15 min before proceeding to compound cleavage.Compound Cleavage: Place the beads into a 700 μL Eppendorf tube. Add 100μL of freshly-prepared 5% (HF/py)/THF solution (v/v). Set the tube toshake for 90 min at room temperature on a tabletop Eppendorf shaker.Quench HF by adding 200 μL TMSOMe to the tube. Set the tube to shake for30 min at room temperature on a tabletop Eppendorf shaker. Collect thefiltrate (if desired) and wash the resin: 3×5 min CH₂Cl₂, 3×5 min THF,and 3×5 min CH₂Cl₂. Dry under house vacuum for at least 1 h beforedecoding.

Representative Bead Decoding Procedure. Place one bead into anautosampler glass sample insert with the aid of tweezers. A 0.24 Msolution of CAN in 5:1 THF/H₂O is prepared (132 mg CAN/0.83 mL dry,degassed THF+0.17 mL doubly-distilled H₂O) in an oven-dried vial. Thissolution should be prepared immediately before use. Add 5 μL of the CANsolution to the glass autosampler insert. Add 8 μL of dry decane to theglass insert and then centrifuge the insert in a Micro-Centrifuge toseparate the two layers. Place the insert in an autosampler vial and captightly. Seal with Parafilm, and heat the glass insert at 37° C. for 21h (in a standard laboratory incubator). Allow the sample to cool to roomtemperature, and remove the glass insert from the autosampler vial.Sonicate the insert for 1-10 min. Centrifuge the insert again in theMicro-Centrifuge. Use a 200 μL Pipetman equipped with a gel-loading tipto remove the top decane layer and transfer it to a new GC autosamplerglass insert. (After heating overnight, the CAN layer will be colorless,so caution must be used to not contaminate the decane layer with CAN intransfer.) Prepare a 1:1 BSA/decane solution in an oven-dried vial. Thissolution should be prepared immediately before use. Add 1.0 μL of thisBSA solution to the decane layer in the GC insert. Spin down the insertin the Microfuge for 30-40 sec to ensure efficient mixing of the BSAsolution with the sample. Place the insert in an autosampler vial, captightly, and store at 0° C. until GC analysis. TABLE 5 Binary decodingdata from GC and LC/MS analysis of 108 beads fromdihydropyrancarboxamide library 12. (BB = building block). GC and MSdata for bead #105 could not be correlated. Tag Tag Tag Tag Tag Tag TagTag Tag Expected Bead # 2B 4B 1A 2A 3A 4A 5A 6A 7A BB1 BB2 BB3 massObserved mass 1 0 1 1 0 1 1 0 0 0 H F A 572 M + H 2 1 0 0 1 0 1 1 0 0 GJ A 539 M + H 3 0 1 1 0 1 1 0 0 0 H F B 552 M + H 4 1 1 0 0 0 0 0 1 0 BD B 435 M + H 5 1 0 0 1 1 1 0 0 0 G F C 447 M + H 6 0 0 0 1 1 0 1 0 0 DG C 447 M + H 7 1 1 0 0 1 0 0 0 0 E A D 441 M + H 8 1 0 0 1 1 0 0 0 0 GA D 401 M + H 9 0 0 0 1 0 0 0 1 0 D D E 514 M + H 10 0 1 0 0 0 0 0 0 1 CE E 500 M + H 11 1 0 0 1 1 0 1 0 0 G G F 473 M + H 12 1 1 0 0 1 1 0 0 0E F F 479 M + H 13 1 1 0 0 1 0 1 0 0 E G G 592 M + H 14 1 1 0 0 0 0 0 01 E E G 596 M + H 15 0 0 0 1 0 1 1 0 0 D J H 540 M − EtOH 16 1 0 0 0 1 00 1 0 A H H 497 M + H 17 0 1 0 0 1 1 0 0 0 B F I 582 M + H 18 0 0 0 1 10 1 0 0 D G I 614 M − BB1_D 19 0 1 0 0 0 1 1 0 0 B J J 640 M + H 20 0 01 0 0 1 1 0 0 C J J 638 M + H 21 1 0 0 0 0 0 0 1 0 A D K 563 M + H 22 00 1 0 1 0 1 0 0 C G K 559 M − EtOH 23 1 0 0 0 1 1 0 0 0 A F L 497 M + H24 0 1 0 0 0 1 1 0 0 B J L 617 M + H 25 0 1 1 0 0 0 1 0 0 H C M 694 M +H 26 0 0 0 1 0 1 1 0 0 D J M 545 M + H 27 1 0 0 1 0 1 1 0 0 G J N 540M + H 28 0 1 1 0 0 0 1 0 0 H C N 655 M + H 29 1 0 0 0 0 1 0 0 0 A B O383 M + H 30 0 0 0 1 0 0 1 0 0 D C O 485 M − BB1_D 31 0 1 0 0 0 1 1 0 0B J P 607 M + MeOH—H₂O 32 1 1 0 0 1 0 0 0 1 E I P 637 M + MeOH—H₂O 33 10 0 1 1 0 0 1 0 G H Q 644 M + H 34 1 0 1 0 1 0 0 1 0 F H Q 684 M + H 351 0 0 1 1 0 0 0 0 G A R 413 M + H 36 0 0 0 1 1 1 0 0 0 D F R 419 M −EtOH 37 0 1 1 0 0 1 1 0 0 H J S 1049 M + H 38 0 1 1 0 1 1 0 0 0 H F S945 M + H 39 0 0 1 0 1 1 0 0 0 C F T 513 M + H 40 0 0 1 0 0 1 1 0 0 C JT 617 M + H 41 0 0 0 1 1 0 0 0 1 D I U 469 M − EtOH 42 1 0 1 0 1 0 1 0 0F G U 515 M − C₄O₂H₈ 43 0 0 0 1 0 0 1 0 0 D C V 558 M − EtOH 44 0 1 0 00 0 1 0 0 B C V 560 M + H 45 1 0 1 0 1 0 0 0 1 F I W 505 M − C₄O₂H₈ 46 10 0 1 0 1 1 0 0 G J W 507 M + H 47 0 0 1 0 1 0 0 0 1 C I X 576 M − EtOH48 1 0 1 0 0 0 0 0 1 F E X 626 M + H 49 1 0 0 0 1 0 0 0 1 A I Y 413 M +H 50 0 0 1 0 0 1 1 0 0 C J Y 469 M + H 51 1 0 0 1 0 0 1 0 1 G D A 487M + H 52 0 0 0 1 1 0 1 1 0 D I A 463 M − EtOH 53 1 0 0 1 0 1 0 1 0 G A B375 M − H₂O 54 0 1 1 0 0 1 0 1 0 H A B 512 M + H 55 0 0 1 0 0 1 0 1 0 HA C 544 M − MeOH 56 0 0 0 1 1 1 0 0 1 D H C 488 M + H 57 0 0 1 0 1 1 0 10 F G D 515 M − EtOH 58 1 0 0 0 0 0 1 0 1 A D D 445 M + H 59 0 0 0 1 0 10 0 1 D B E 456 M + H 60 1 0 0 1 0 1 0 0 1 G B E 490 M + H 61 1 0 0 1 01 0 0 1 G B F 433 M + H 62 0 1 1 0 0 0 0 1 1 H E F 614 M + H 63 1 0 0 01 0 1 0 1 A J G 574 M + H 64 0 0 0 1 1 1 0 0 1 D H G 559 M + H 65 0 0 01 1 0 1 1 0 D I H 498 M − EtOH 66 0 0 1 0 1 0 1 0 1 C J H 540 M + H 67 01 0 0 0 1 0 0 1 B B I 576 M + H 68 0 1 1 0 0 0 0 1 1 D E I 618 M + H 691 0 0 1 1 1 0 1 0 G G J 602 M + H 70 1 0 0 1 0 1 0 1 0 G A J 528 M + H71 1 1 0 0 0 0 1 0 1 E D K 651 M + H 72 1 0 1 0 0 0 0 1 1 F E K 637 M +H 73 1 0 1 0 1 1 1 0 0 F F L 585 M + H 74 1 0 0 1 0 0 0 1 1 G E L 583M + H 75 1 0 1 0 1 1 0 0 1 F H M 590 M + H 76 1 0 1 0 0 0 0 1 1 F E M553 M − C₄O₂H₈ 77 0 0 1 0 1 0 1 0 1 C J N 506 M + H 78 1 0 0 1 0 0 1 1 0G C N 518 M + H 79 1 0 0 0 0 0 1 1 0 E C O 559 M + H 80 1 1 0 0 1 1 1 00 E F O 477 M + H 81 1 0 0 1 1 1 0 1 0 G G P 569 M + MeOH—H₂O 82 0 1 1 00 0 1 1 0 H C P 754 M + H 83 0 0 0 1 1 1 0 1 0 D G Q 569 M − EtOH 84 1 00 1 1 1 0 1 0 G G Q 603 M − H₂O 85 1 0 0 0 1 0 1 1 0 A I R 467 M + H 860 0 1 0 0 0 0 1 1 C E R 457 M − EtOH 87 0 1 0 0 1 1 0 1 0 B G S 810 M +H 88 1 0 0 0 1 1 0 1 0 A G S 794 M + H 89 1 0 1 0 1 1 0 1 0 F G T 621 M− C₄O₂H₈ 90 1 0 0 1 0 0 1 1 0 G C T 629 M + H 91 1 0 1 0 0 0 1 0 1 F D U533 M − C₄O₂H₈ 92 0 0 1 0 1 0 1 0 1 C J U 511 M + H 93 0 0 1 0 0 1 0 1 0C A V 436 M − EtOH 94 0 0 0 1 0 0 1 1 0 D C V 558 M + H 95 0 1 1 0 0 1 01 0 H A W 500 M + H 96 0 0 1 0 1 0 1 0 1 C J W 473 M + H 97 1 1 0 0 0 00 1 1 E E X 626 M + H 98 0 0 1 0 1 1 1 0 0 C F X 514 M + H 99 0 0 1 0 11 1 0 0 C F Y 365 M + H 100 0 0 0 1 1 0 1 1 0 D I Y 427 M − EtOH 101 1 00 0 1 0 0 0 0 A A 298 M + Na 102 1 0 1 0 1 0 0 0 1 F I 488 M − EtOH 1030 0 0 1 1 1 0 0 1 D H 427 M − EtOH 104 0 1 0 0 0 1 0 0 1 B B 348 M + H105 1 0 1 0 1 0 1 0 0 F G 420 M − C₄O₂H₈ 106 1 0 1 0 1 0 0 0 0 F A 346M + Na 107 1 1 0 0 0 0 1 0 1 E D 438 M − EtOH 108 0 1 0 0 1 1 0 1 0 B G348 M + H

Example 3 Biological Testing

1. Discussion of Methodology

Cell and Protein Based Screens

It will be appreciated that the small molecule compounds of the presentinvention may be screened in any of a variety of biological assays. Forexample, cell-based assays may be employed (see FIG. 11). Suchcell-based assays generally involve contacting a cell with a compoundand detecting any of a number of events, such as binding of the compoundto the cell, initiation of a biochemical pathway or physiological changein the cell, changes in cell morphology, initiation or blockage of thecell cycle etc.

In but one example, once synthesized, the compounds may be arrayed in384-well plates by a robotic 384 pin arrayer, as shown in FIG. 23, andassayed for their ability to bind to a particular cell type present inthe well. Detection can be carried out, for example, by detecting a tagthat is attached to the small molecule. Alternatively, the smallmolecule may be detected by using a second molecule that has a tag, thesecond molecule specifically binding the small molecule, e.g., a taggedantibody specific to the small molecule.

Alternatively or additionally, inventive compounds may be studied insuch assays. In such assays, the compounds are bound to a solid supportand then contacted with a protein of interest. The presence or absenceof binding between the compound and the protein is then detected. Incertain cases, the protein itself is tagged with a molecule that can bedetected, e.g., with a fluorescent molecule. Alternatively, the proteinis detected by utilizing any immunoassay, such as the ELISA.

For example, a process known as small molecule printing (see, forexample, U.S. Ser. No. 09/567,910, filed May 10, 2000, the entirecontents of which are hereby incorporated by reference), may be utilizedto screen proteins that interact with the library compounds. First, asplit pool library is arrayed onto beads. The compounds are then cleavedfrom the beads and prepared in a standard stock solution, such as DMSO.The compounds are then arrayed onto a 384-well stock plate. Next, thecompounds are printed onto glass slides, e.g., a glass microscopeslides, and the slides are probed with a tagged ligand, e.g., a taggedprotein of interest. Binding between a compound and the ligand is thendetected by any available means appropriate to the tag being utilized,e.g., via fluorescence. (See FIGS. 24 and 25).

It will be appreciated that any of the general assay methods describedabove, as well as other assays known in the art, may identifydihydropyrancarboxamide-like molecules having certain biologicalproperties. Described below are assays that examples of assays that wereused to screen the inventive library of compounds, and that helpedidentify library members that exhibited certain biological activity(e.g., BdrU incorporation, Genistein suppressor activity and Eg5inhibition).

As discussed above, in certain embodiments, the inventive library isprepared by three diversity-generating steps, the first two of whichwere encoded with chloroaromatic tags, as described in Example 2 above.As the final diversity-generating step was not chemically encoded, thelibrary was prepared as 54 separate portions of dry resin (9) totalingthree theoretical copies of 4320 stereochemically and structurallydistinct compounds (10) (see Scheme 4).

Cleavage and Elution of a Diversity Set of Dihydropyrancarboxamides

A robotic cleavage and elution protocol was used to deliver actuallibrary members from the inventive library into chemical genetic assays.As described in Scheme 5, an encoded, split-pool library of 4320dihydropyrancarboxamides (10) (an exemplary synthesis of which isdescribed herein. See also, R. A. Stavenger, S. L. Schreiber, Asymmetriccatalysis in diversity-oriented organic synthesis: enantioselectivesynthesis of 4320 encoded and spatially segregateddihydropyrancarboxamides, Angew. Chem. Int. Ed. 2001, 40:3417-3421).

Briefly, as discussed above, in one embodiment, a method for preparingthis library comprises three diversity-generating steps, the first twoof which were encoded with chloroaromatic tags as described in H. E.Blackwell, L. Pérez, R. A. Stavenger, J. A. Tallarico, E. Cope-Eatough,S. L. Schreiber, M. A. Foley, “A one-bead, one-stock solution approachto chemical genetics, part 1”, Chem. Biol. 2001, 8:1167-1182. As thefinal diversity-generating step was not chemically encoded, we acquiredthis library as 54 separate portions of dry resin (9) totaling threetheoretical copies of 4320 stereochemically and structurally distinctcompounds (10). We first exposed 324 individual beads, six from each ofthe 54 separate portions of 9, to our manual ‘best practices’ cleavageand elution conditions (Scheme 5) in a single microtiter plate. In thiscase, compounds were eluted directly into DMF to prepare a diversityplate of stock solutions (plate 0) amenable to small molecule printing.Glass microscope slides were activated for covalent attachment ofalcohols, and compounds (10) from the 320 stock solutions were printedaccording to a method described in U.S. patent application Ser. No.09/567,910 (see also, P. J. Hergenrother, K. M. Depew, S. L. Schreiber,Small molecule microarrays: covalent attachment and screening ofalcohol-containing small molecules on glass slides, J. Am. Chem. Soc.122 (2000) 7849-7850).

To test the availability of 10 to a protein-binding assay, we probed thesmall molecule microarray with purified Cy5-labeled (His)6-FKBP12 (SeeG. MacBeath, A. N. Koehler, S. L. Schreiber, Printing small molecules asmicroarrays and detecting protein-ligand interactions en masse, J. Am.Chem. Soc. 121 (1999) 7967-7968). As a positive control forprotein-ligand interaction, AP1497 was included on the slide by addingit in DMF solution to an empty well of the stock plate (See, forexample, D. A. Holt, J. I. Luengo, D. S. Yamashita, H.-J. Oh, A. L.Konialian, H.-K. Yen, L. W. Rozamus, M. Brandt, M. J. Bossard, M. A.Levy, D. S. Eggleston, J. Liang, L. W. Schultz, T. J. Stout, J. Clardy,Design, synthesis, and kinetic evaluation of high-a§nity FKBP ligandsand the X-ray crystal structures of their complexes with FKBP12, J. Am.Chem. Soc. 115 (1993) 9925-9938; and J. F. Amara, T. Clackson, V. M.Rivera, T. Guo, T. Keenan, S. Natesan, R. Pollock, W. Yang, N. L.Courage, D. A. Holt, M. Gilman, A versatile synthetic dimerizer for theregulation of protein-protein interactions, Proc. Natl. Acad. Sci. USA94 (1997) 10618-10623). Following incubation, the slide was washed andscanned for the presence of a Cy5 fluorescence signal (See G. MacBeath,A. N. Koehler, S. L. Schreiber, Printing small molecules as microarraysand detecting protein-ligand interactions en masse, J. Am. Chem. Soc.121 (1999) 7967-7968), which appeared both at the AP1497 control spots(data not shown) and at spots corresponding to a member of 10 (FIG. 13(a)). The bead corresponding to the novel FKBP12-binding entity wassubjected to the optimized bead decoding protocol described in Example 2herein. Using this procedure, we were able unambiguously to determinethe structure (FIG. 13 (b)) of this ‘hit’ (11) in a protein-bindingassay, as was subsequently confirmed by tandem liquidchromatography/mass spectroscopy (LC/MS).

Formatting and Assaying of Representative Dihydropyrancarboxamides

To apply the robotic process to a fraction of resin 9, we arrayed 128beads from each of three separate portions of 9 into a single 384-wellmicrotiter plate. These beads were subjected to robotic cleavage andCH₃CN elution as described earlier to prepare a ‘mother plate’ (plate 1)containing 384 members of 10. Subsequently, the ‘mother plate’ wasmapped into six ‘daughter plates’ by volumetric transfer using thesyringe-array robot. ‘Daughter plates’ were prepared for cell-basedassays^([1,2]) (50% of stock solution), HPLC analysis (25%), LC/MSanalysis (10%), small molecule printing^([3,4]) (2×5%), and stocksolution decoding (5%) [(1) T. U. Mayer, T. M. Kapoor, S. J. Haggarty,R. W. King, S. L. Schreiber, T. J. Mitchison, Small molecule inhibitorof mitotic spindle bipolarity identified in a phenotype-based screen,Science 286 (1999) 971-974; (2) B. R. Stockwell, S. J. Haggarty, S. L.Schreiber, High-throughput screening of small molecules in miniaturizedmammalian cell-based assays involving post-translational modifications,Chem. Biol. 6 (1999) 71-83; (3) G. MacBeath, A. N. Koehler, S. L.Schreiber, Printing small molecules as microarrays and detectingprotein-ligand interactions en masse, J. Am. Chem. Soc. 121 (1999)7967-7968; and (4) P. J. Hergenrother, K. M. Depew, S. L. Schreiber,Small molecule microarrays: covalent attachment and screening ofalcohol-containing small molecules on glass slides, J. Am. Chem. Soc.122 (2000) 7849-7850]. In each case, the CH₃CN solution was evaporatedfollowing volumetric transfer so that each copy could be resuspended inthe solvent most appropriate to its use. In particular, DMSO was used toresuspend the ‘daughter plate’ for cell-based assays and DMF was used toresuspend the ‘daughter plate’ for small molecule printing. The platecontaining the beads was also stored, but due to the success of stocksolution decoding [H. E. Blackwell, L. Pérez, S. L. Schreiber, Decodingproducts of diversity pathways from stock solutions derived from singlepolymeric macrobeads, Angew. Chem. Int. Ed. 40 (2001) 3421-3425], andthe difficulties associated with maintaining positional integrity withinplates of beads, formatting a ‘daughter plate’ explicitly destined forstructure determination has become the standard in our libraryrealization process.

Both plates of stock solutions (10) were used in phenotypic assays. Inparticular, we exposed living human A549 lung carcinoma cells to 708(324+384) stock solutions under two different assay conditions. Theseexperiments were performed with a hand-held pin-transfer tool, thoughour complete technology platform includes a pin-transfer robot capableof mapping into multiple microtiter plates. In certain embodiments,cultured cells exposed to 5-bromodeoxyuridine (BrdU) will incorporatethis base analog into their DNA when actively dividing, and thisincorporation can be detected by cytoblot assay using antibodiesdirected against BrdU (B. R. Stockwell, S. J. Haggarty, S. L. Schreiber,High-throughput screening of small molecules in miniaturized mammaliancell-based assays involving post-translational modifications, Chem.Biol. 6 (1999) 71-83). First, to determine if any stock solution of 10inhibits BrdU incorporation, we transferred ˜100 nl of each stocksolution into individual assay wells containing A549 cells activelygrowing in the presence of 1% fetal bovine serum. Second, we exposedA549 cells to ˜100 nl of each stock solution, and simultaneouslychallenged the cells with 100 μM genistein, a broad-spectrum proteintyrosine kinase inhibitor (T. Akiyama, J. Ishida, S, Nakagawa, H.Ogawara, S. Watanabe, N. Itoh, M. Shibuya, Y. Fukami, Genistein, aspecific inhibitor of tyrosine-specific protein kinases, J. Biol. Chem.262 (1987) 5592-5595). Under the latter conditions, BrdU incorporation,again judged by cytoblot assay, is impaired (For a discussion of thecytoblot assay technology, see U.S. patent application Ser. No.09/361,576; and B. R. Stockwell, S. J. Haggarty, S. L. Schreiber,High-throughput screening of small molecules in miniaturized mammaliancell-based assays involving post-translational modifications, Chem.Biol. 6 (1999) 71-83). Thus, ‘hits’ in the former assay are detected asa loss of signal in a high-signal array (FIG. 14 (a)), while ‘hits’ inthe latter assay are detected as a gain of signal in a lowsignal array(FIG. 14 (b)). The latter assay is referred to as a genistein suppressorscreen, as we are seeking a member of 10 that can suppress the abilityof genistein to inhibit BrdU incorporation.

For each of these assays, aliquots from each of the two plates (10) wereexposed to cells in duplicate to ensure the fidelity of the results.Compounds were scored as ‘hits’ only if they scored strongly in bothreplicates of a given experiment. From plate 0, 11 compounds scored asinhibitors of BrdU incorporation, while 10 compounds scored assuppressors of the action of genistein. From plate 1, 12 compoundsscored as inhibitors of BrdU incorporation, while nine compounds scoredas suppressors of the action of genistein. It is interesting thatroughly the same number of first-pass ‘hits’ were identified on eachplate, despite the difference in diversity between the two collections.This finding may reflect the fact that assay results were tabulated byvisual scoring of photographic film, but is not limited to suchdetection methods. Conversely, in the case of an FKBP12-binding assayusing microarrayed compounds, plate 1 produced no ‘hits’ (data notshown).

To ensure that we can obtain exact structural information on the ‘hits’found in these experiments, we performed either bead decoding [H. E.Blackwell, L. Pérez, R. A. Stavenger, J. A. Tallarico, E. Cope-Eatough,S. L. Schreiber, M. A. Foley, A one-bead, one-stock solution approach tochemical genetics, part 1, Chem. Biol. 8 (2001) 1167-1182] or stocksolution decoding [H. E. Blackwell, L. Pérez, S. L. Schreiber, Decodingproducts of diversity pathways from stock solutions derived from singlepolymeric macrobeads, Angew. Chem. Int. Ed. 40 (2001) 3421-3425] on all42 compounds scoring as positive in either assay. Decoding results werecompared with LC/MS results for each sample to verify that a compound ofthe correct mass was present. In all but nine cases, LC traces revealeda single clean peak, and for each of the 42 ‘hits’, a parent ion orfragment matching the proposed structure was observed by MS. Thus, wewere able to decode and confirm the structure (FIG. 14 (c)) of each‘hit’ detected in either the BrdU or the genistein suppressor cytoblotassay.

The library was also screened for Eg5 inhibitory activity. The followinglibrary member was found to inhibit Eg5 (FIG. 15):

From a statistical perspective, the library of dihydropyrancarboxamides(9) was fully encoded, either chemically using chloroaromatic tags(first two diversity-generating steps), or positionally by inclusioninto one of 54 pools of resin (third diversity-generating step). Ourcollection of decoded ‘hits’ was analyzed to assign statisticalsignificance to a process of ‘codon’ selection, by a given assay, ofparticular encoded events (or combinations of encoded events) during thechemical history of the library. The formal details of this analyticalprocess will be reported once applied to the entire collection of 4320dihydropyrancarboxamides (9). One immediate consequence is that aconsensus set of structures corresponding to a particular assay activityneed not be limited to individual structures that scored as ‘hits’ inthe assay. For example, if two codons corresponding to building blocksfrom two different diversity-generating steps were each stronglyselected by a given assay, one might predict that a compoundincorporating both moieties would yield higher potency in that assay. Inthe absence of additional information, we would predict such a consensusstructure even if the exact compound in question was not present in theinitial screen. Alternatively, if the assay in question selected againstthis particular combination of codons, we would uncover this ‘forbidden’combination, even if each codon alone was frequently observed amongstructures scoring as ‘hits’. Traditionally, structure-activityrelationships are determined by processes ranging from an intuitiveviewing of ‘hit’ structures to a comparison of ‘hits’ on the basis ofexisting quantitative molecular descriptors (each based on somearbitrary metric). Our analysis introduces a novel approach, whereby werequire no structural information in advance of defining significantbiological activity. Rather, we allow the biological system under studyto dictate the requirements for its activity. Such analysis illustratesthe power of annotation screens to inform chemistry, through thetechnology platform, in ways that can influence planning steps in futurediversity-oriented syntheses.

CONCLUSION

A technology platform aimed at advancing chemical genetics was appliedto the identification of novel dihydropyrancarboxamides with certainbiological activities. The platform encompasses an optimized procedurefor compound cleavage and elution from large PS beads, a novel beadarraying method, and robotic implementation of library formatting, theprocess by which small molecules from diversity-oriented syntheses aremade accessible to chemical genetic assays. We validated this approachby successfully synthesizing, encoding, and formatting a split-poollibrary of dihydropyrancarboxamides (9). It is important to note thatoptimization of the library formatting process occurred independently ofthe development of chemistry required to synthesize the library. Rather,optimization of the formatting process used generic model compounds toestablish parameters, while formatting the split-pool library used theoutput of the optimization as a general, or ‘best practices’, method forlibrary realization.

By exposing each member of a diversity-oriented synthesis to multiplephenotypic and proteomic assays, we can annotate each compound in thecollection in a way that is complementary to other methods of smallmolecule characterization, such as MS and NMR. Statistical analysis ofthe biological performance of an encoded collection of small moleculesallows us to inform further synthetic efforts (e.g. scaled synthesis ofsubset libraries based on primary screening data) in ways notnecessarily available by traditional structure activity analyses.Annotation screening is a term we use to describe the generation ofmultiple datasets by comprehensive screening of such libraries over arange of biological outcomes. The analysis of data resulting fromannotation screening comprises both the challenge and the promise ofchemical genetic research.

2. Experimental

Materials and Methods

Model Resin Preparation

2-Naphthaleneethanol (6), K-methyl-2-naphthalenemethanol (7), and2-naphthol (8) were obtained commercially (Sigma-Aldrich) and driedazeotropically prior to the loading reaction. Resin 1 was a generousgift of Max Narovlyansky and Dr. John A. Tallarico, and contains ˜200mmol Si/bead calculated based on elemental analysis, assuming that 550μm is the average bead size in a population of beads pre-sized at500-600 μm. Loading reactions were performed in fritted polypropylenePD-10 columns (Amersham Pharmacia Biotech) and agitated by rocking on aLabquake™ (Barnstead Thermolyne) shaker.

Resin samples were washed on a Vac-Man® vacuum manifold (Promega) fittedwith nylon stopcocks (Bio-Rad). HPLC-grade reaction solvents (J. T.Baker) were purified by passage through two solvent columns prior touse. Et₃N and 2,6-lutidene were distilled over calcium hydride. Inloading reactions, bromostyrene-copolymerized beads were added to aPD-10 (Amersham Pharmacia Biotech) column, which was capped with aseptum and plastic stopcock and flushed with Ar. After swelling withCH₂Cl₂ (10 ml), a 2.5% (v/v) solution of TMSCl in CH₂Cl₂ was added. Thebeads were suspended for 15 min and filtered with Ar pressure. The beadswere washed with CH₂Cl₂ (3×2 min), then suspended in a solution of TfOH(6 eq.) in CH₂Cl₂ for 15 min, during which time Ar was bubbled gentlythrough the reaction via a syringe. Next, the beads were rinsed withCH₂Cl₂ (3×2 min) under Ar and suspended in CH₂Cl₂. Freshly distilled2,6-lutidine (8 eq.) and model alcohol 6, 7, or 8 (3 eq.) weresuccessively added. The tube was capped and sealed to stand for 18 h atambient temperature, after which the beads were filtered and rinsed withCH₂Cl₂ (4×3 min) and dried under house vacuum.

Cleavage and Quenching

Commercially available HF/py (Sigma-Aldrich) is approximately a 7:3mixture of HF and pyridine, which was buffered with additional pyridinein THF solution. In manual experiments, beads were transferredindividually by forceps to wells of 384-well microtiter plates(Genetix). Cleavage and quenching reagents, as well as elution solvents,were added by a P20 single-channel pipettor (Gilson). Data from 19F NMRexperiments were obtained at 470.169 MHz on a Varian (Varian, Inc.,http://www.varianinc.com/) AS500 (nt=128). To avoid etching of the NMRtube by HF/py solutions, samples were placed in a PTFE-FEP NMR tubeliner (Wilmad-LabGlass).

HPLC Quantitation

HPLC analysis was carried out using a ThermoSeparation Products(Thermo-Finnigan) instrument with a PC0100 system controller andassociated software. All samples were run on a Hypersil C18mini-pharmaceutical column (The Nest Group) using a flow rate of 3ml/min, an 80 s gradient of 0-99.9% CH₃CN in water/0.1% trifluoroaceticacid/0.1% methanol, and diode array detection. Single peaks at 224 nmabsorbance were characteristic of compounds 6 (rt=1.54 min), 7 (rt=1.54min), and 8 (rt-1.49 min). To establish boundary conditions fordetection of cleaved compounds by HPLC, standard curves were determinedusing pure samples of 6-8. Mock cleavage reactions (no HF present, butotherwise treated as described in the text) were carried out on resins3-5 to determine the experimental noise for our HPLC detection method.

Robotic Implementation

Before bead arraying, 384-well plates (Genetix) were pre-wetted using aMultidrop 384 (Thermo-Labsystems) to dispense solvent. HF/py solutionswere delivered using an Ivek multiplex controller module with linearactuator pump module (Ivek Corporation, http://www.ivek.com/) coupled toan ADM-661 automatic dispensing system with TruPath 300 controllermodule (Creative Automation, http://www.creativedispensing.com/), andfully contained within a Captair ductless fume hood with recirculatingair filtration system (Captair LabX, http://www.erlab-dfs.com/).Automated plate handling was carried out by Twister Universal microplatehandlers (Zymark Corporation, http://www.zymark.com/). Evaporation ofquenched reaction mixtures was done using a GeneVac HT4 Atlas evaporatorwith VC3000D vapor condenser (GeneVac Technologies,http://www.genevac.co.uk/). Elution of compounds from beads into 100μl/well ‘mother plates’ (Marsh), as well as formatting of 50 μl/well‘daughter plates’ (Genetix), was done with a Hydra Microdispenser 384(Robbins Scientific Corporation, http://www.robsci.com/).

Small Molecule Microarrays

Small molecules were printed as described in P. J. Hergenrother, K. M.Depew, S. L. Schreiber, Small molecule microarrays: covalent attachmentand screening of alcohol-containing small molecules on glass slides, J.Am. Chem. Soc. 122 (2000) 7849-7850, either with a microarray robotbuilt as described by Dr. Pat O. Brown(http://cmgm.stanford.edu/pbrown/mguide/), or with an Omni-Grid™multi-axis robot (GeneMachines, http://www.genemachines.com/). Briefly,slides were activated for covalent attachment of alcohols as describedpreviously. Standard microscope slides (VWR, 48300-036) were cleaned inpiranha solution (70:30 v/v solution of concentrated H₂SO₄ and 30% H₂O₂)for 16 hours at room temperature. The slides were washed extensively inddH₂O and kept in water until use. To convert to a silyl chloridesurface, the slides were removed from water and dried by centrifugation.The slides were then immersed in a solution of dry THF containing 1%SOCl₂ and 0.1% DMF. The slides were incubated in this activatingsolution for 4 hours at room temperature. The slides were then removed,washed briefly in THF, and then placed onto the encased microarrayerplatform under argon. Small molecules were printed as describedpreviously. Printing was carried out using a microarraying robot,constructed in this laboratory by Dr. James Hardwick and Dr. Jeff Tongaccording to directions provided by Dr. Pat Brown(http://cmgm.stanford.edu/pbrown/mguide/). The microarrayer typicallywithdraws 250 mL from a 384-well (or 96-well) plate and repetitivelydelivers 1 mL to defined locations on a series of activated slides. Thepins were washed for 8 seconds in acetone and dried under vacuum for 8seconds in between each sample. The arrayer was instructed to print thesamples described here approximately 500 μm apart. Following printing,the slides were allowed to stand at ambient temperature for 12 hours.The slides were then washed for 2 hours in DMF, 1 hour in THF, and 1hour in ethanol. Slides were dried by centrifugation and were at roomtemperature under vacuum until use.

(His)6-FKBP12 was purified to homogeneity as described in G. MacBeath,A. N. Koehler, S. L. Schreiber, Printing small molecules as microarraysand detecting protein-ligand interactions en masse, J. Am. Chem. Soc.121 (1999) 7967-7968. Cy5-labeled protein was prepared using FluoroLink™monofunctional reactive dye (Amersham Pharmacia Biotech) according tothe manufacturer's protocol. Fluorescence detection of binding eventswas monitored using an ArrayWoRx biochip reader (Applied Precision,http://www.api.com/).

N-terminal His-tagged FKBP12 was expressed using the T5 expressionplasmid pQE-30-FKBP12 (3757 bp) in M15-[pREP4] (Qiagen) purified tohomogeneity as described previously. A starter culture was prepared byinoculating 5 mL LB medium supplemented with 100 μg/mL sodium ampicillinand 50 μg/mL kanamycin from a single colony and grown for 16 hours at37° C. The cells were subcultured into 500 mL of the same medium at aninitial OD₆₀₀ of 0.1. The culture was grown at 37° C. up to an OD₆₀₀ of0.8. The culture was cooled to room temperature and isopropyl1-thio-β-D-galactopyranoside (IPTG) was added to a final concentrationof 1 mM. After a 16 hour induction at 30° C., the cells were harvestedand frozen at −80° C. for 24 hours. The cell pellet was resuspended in20 mL of PBS buffer supplemented with 10% (v/v) glycerol and a proteaseinhibitor cocktail mini-tablet (Boerhinger Mannheim). Cells were lysedby addition of 1 mg lysozyme per gram of wet cell pellet. The suspensionwas incubated on ice for 1 hour and followed by a 4 minute incubation at37° C. with gentle mixing. The lysate was then kept on ice for 10minutes. The lysate was clarified by centrifugation (28,000 g, 30minutes, 4° C.) and loaded onto a column packed with 5 mL of Ni-NTA(Qiagen) that had been equilibrated in PBS. The column was washed with50 mL of PBS buffer containing 10 mM imidazole. Protein bound to thecolumn was eluted with PBS buffer containing 250 mM imidazole. Thesample was dialyzed against PBS at 4° C. Cy5-labeled (His)6-FKBP12 wasprepared using FluoroLink™ monofunctional reactive dye (AmershamPharmacia Biotech) according to the manufacturer's protocol. Slides wereblocked for 1 hour by incubation with PBST (PBS buffer containing 0.1%Tween-20) containing 3% BSA. After a brief rinse with PBST,fluorescently labeled protein was added a concentration of 1 μg/mL inPBST supplemented with 1% BSA. Slides were incubated with labeledprotein for 30 minutes at room temperature. Slides were then washed inPBST for 3 minutes three times and dried by centrifugation. Slides werethen scanned using an ArrayWoRx slide scanner (Applied Precision) at aresolution of 5 μm per pixel. The following filter sets were employed:Cy5 excitation/emission (1 second exposure) and Cy3 excitation/emission(1 second exposure).

Cell-Based Assays

Transfer of stock solutions of 10 into assay plates (Nunc) was doneusing a VP386 384-pin MultiBlot™ replicator (VpP Scientific,http://www.vp-scientific.com/). Cell culture methods and the BrdU assayprotocol were carried out as described in B. R. Stockwell, S. J.Haggarty, S. L. Schreiber, High-throughput screening of small moleculesin miniaturized mammalian cell-based assays involving post-translationalmodifications, Chem. Biol., 6:71-83, 1999. Detection of assay resultswas carried out using X-oMAT AR film (Kodak), and multiplicativeoverlays of digitally scanned replicate films were prepared usingPhotoshop 5.0 (Adobe Systems). The Multidrop 384 liquid dispenser(Labsystems) was used for all liquid additions, and a 24-channel wand(V&P Scientific) attached to a house vacuum source was used for allliquid aspirations. Two thousand A549 cells were seeded per well of a384-well plate (Nalge Nunc, white, tissue culture treated) in Dulbecco'smodified Eagle medium (DMEM) supplemented with 10% fetal bovine serum(FBS). Immediately upon seeding, 50 mL compound from the RAScombinatorial library was pin-transferred, one compound per well, from a5-mM stock solution in DMSO to a final concentration of 5 μM. After 24hours at 37° C. with 5% CO₂, 10 μL of a 10× stock of bromodeoxyuridine(BrdU) in DMEM+10% FBS was added, for a final concentration of 10 μMBrdU. The cells were incubated for 4 hours at 37° C. with 5% CO₂, cooledon ice for 15 minutes, and fixed in 50 μL 70% ethanol/30% phosphatebuffered saline (PBS). All subsequent steps were performed at 4° C.Cells were washed with 90 μL cold PBS, incubated in 25 μL 2 M HCl/0.5%Tween-20 in ddH₂O for 20 minutes at room temperature, and incubatedinstantly in 90 μL 10% 2 M NaOH/90% Hanks Buffered Salt Solution (HBSS;Gibco BRL). Cells were washed twice with 90 μL HBSS and blocked with 75μL PBSTB (PBS, 0.1% Tween-20, 3% bovine serum albumin). Subsequently, 20μL antibody solution, consisting of 0.5 μg/mL mouse anti-BrdU(Pharmingen), diluted 1:1000 in PBSTB, and anti-mouse IgG conjugated tohorseradish peroxidase (HRP; Amersham), diluted 1:2000 in PBSTB, wasadded to each well. After overnight incubation at 4° C., wells werewashed twice in 90 μL PBS and detected with 20 μL HRP substrate solution(ECL detection; Amersham). Film (X-OMAT AR; Kodak) was placed on top ofthe plates in a darkroom and developed after one to five minutes with aKodak M35A X-OMAT processor.

Eg5 Inhibition

Cloning and Expression of Eg5 Constructs

Coding regions for the expression of C-terminally His₆-tagged constructsof human Eg5 were generated by polymerase chain reaction using apBluescript template containing full length human Eg5 [23] and thefollowing primers: a common N-terminal primer5′-GCAACGATTAATATGGCGTCGCAGCCAAATTCGTCTGCGAAG and specific C-terminalprimers; 5′-GCAACGCTCGAGTCAGTGATGATGGTGGTGATGCTGATTCACTTCAGGCTTATTCAATAT(hEg5-367H),5′-GCAACGCTCGAGTCAGTGATGATGGTGGTGATGCATGACTCTAAAATTTTCTTCAGAAAT(hEg5-405H),5′-GCAACGCTCGAGTCAGTGATGATGGTGGTGATGTGTAACCCTATTCAGCTCCTCCTCAACAGC(hEg5-437H). The PCR products were ligated into a pRSETa backbone. Eg5protein constructs were expressed and purified as described previously[Woehlke, G., Ruby, A. K., Hart, C. L., Ly, B., Hom-Booher, N. and Vale,R. D. (1997) Microtubule interaction site of the kinesin motor. Cell.90, 207-216]. The Eg5 containing fractions from Superose 6 sizingchromatography were pooled, supplemented with sucrose to 10% (w/v) as acryo-protectant, flash frozen in liquid nitrogen, and stored at −80° C.The concentration of Eg5 was measured using the Edelhoch [Pace, C. N.,Vajdos, F., Fee, L., Grimsley, G. and Gray, T. (1995) How to measure andpredict the molar absorption coefficient of a protein. Protein Science.4(11), 2411-2423] as well as Bradford techniques.

Steady-State Eg5 ATPase Assay

We measured the ATPase activity of Eg5 in vitro using an assay thatcouples the hydrolysis of ATP to the oxidation of NADH [Woehlke, G.,Ruby, A. K., Hart, C. L., Ly, B., Hom-Booher, N. and Vale, R. D. (1997)Microtubule interaction site of the kinesin motor. Cell. 90, 207-216].In the assay, the concentration of ATP remains constant however, thedecrease in NADH fluorescence is a convenient measure of the amount ofATP turned over. Our typical reaction buffer contained 25 mM PotassiumChloride, 25 mM Potassium PIPES (6.90), 2 mM Magnesium Chloride, 1 mMPotassium Phosphoenol Pyruvate, 200 μM di-Potassium NADH, 1 mMDithiothreitol, 10 μM Taxol, 9 U/ml Lactate Dehydrogenase, 1 U/mlPyruvate Kinase and taxol-stabilized microtubules as needed. To measurethe ATPase activity in a reaction, the assay buffer was supplementedwith 1 mM MgCl₂:ATP (1:1), 1 μM microtubules and 40 nM Eg5-367H.Time-points for NADH fluorescence were measured in 384 well black plates(NalgeneNUNC) by a Wallac Victor² 1420 multilabel counter, umbelliferonefilter set (excitation: 355 nm, emission: 420 nm), and the steady-staterate of fluorescence decay was calculated using a linear fit byMicrosoft Excel. The coupling activity of the enzyme system was 100-foldgreater than the Eg5 ATPase activity used in our experiments. Tocalculate IC₅₀ values for enantiomerically pure monastrol in thepresence or absence of microtubules, we fit enzyme velocity as afunction of monastrol concentration to the equation:V=V_(residual)+(V_(inhibited)×IC₅₀)/(IC₅₀+[monastrol]). Enzymevelocities were fit as a function of microtubule or ATP concentrationsat particular monastrol concentration to the equation:V=([substrate]×V_(max))/([substrate]+K_(m)).

Cell Culture Methods

BS—C-1 cells were cultured on glass coverslips as described previously[Cramer L P, Mitchison T J, Theriot J A. (1994) Actin-dependent motileforces and cell motility. Curr. Opin. Cell. Biol. 6(1), 82-6]. AB9Zebrafish cells [Hukreide N A, Joly L, Tsang M, et. al. (1999) Radiationhybrid mapping of the zebrafish genome. Proc. Natl. Acad. Sci. 96,9745-9750] were grown in DMEM at 28° C. in a 5% CO₂ atmosphere onpolylysine coated coverslips. Cells were grown to 50% confluence, rinsedwith warm PBS, and incubated an additional 6 hours in growth mediumsupplemented with 20 mM Potassium HEPES and compound at a final DMSOconcentration of 0.2%. Cells were fixed with 1% formaldehyde in 1× TrisBuffered Saline containing 0.1% Triton X-100 as detergent. Cells wereso-stained with Alexa-488-conjugated goat anti-mouse antibodies, DM-1A,a mouse antibody against α-tubulin (Sigma), and Hoechst dye. We countedmono-astral, mitotic, and interphase cells by visual inspection tocalculate the percentage of monastral cells at each drug concentration.

1. A compound having the structure:

wherein R₁, R₂ and R₄ are each independently hydrogen or an aliphatic,heteroaliphatic, aryl, heteroaryl, alkylaryl or alkylheteroaryl moiety;R₃ is hydrogen or an aliphatic, heteroaliphatic alkylaryl, heteroaryl oralkylheteroaryl moiety; R₅ and R₆ are each independently hydrogen or analiphatic, heteroaliphatic, aryl, heteroaryl, alkylaryl, oralkylheteroaryl moiety, and wherein R₆ and R₇, taken together, may forma cyclic aliphatic, heteroaliphatic, aliphatic(aryl),heteroaliphatic(aryl), aliphatic(heteroaryl) orheteroaliphatic(heteroaryl) moiety, or an aryl or heteroaryl moiety;wherein each of the foregoing aliphatic and heteroaliphatic moieties maybe substituted or unsubstituted, cyclic or acyclic, saturated orunsaturated or linear or branched; and each of the foregoing aryl,heteroaryl, alkylaryl or alkylheteroaryl moieties may be substituted orunsubstituted; and pharmaceutically acceptable derivatives thereof. 2.The compound of claim 1, wherein the compound has the structure (II):

wherein R₁, R₂ and R₄ are each independently hydrogen or an aliphatic,heteroaliphatic, aryl, heteroaryl, alkylaryl or alkylheteroaryl moiety;R₃ is hydrogen or an aliphatic, heteroaliphatic, alkylaryl, heteroarylor alkylheteroaryl moiety; R₅ and R₆ are each independently hydrogen oran aliphatic, heteroaliphatic, aryl, heteroaryl, alkylaryl, oralkylheteroaryl moiety, and wherein R₆ and R₇, taken together, may forma cyclic aliphatic, heteroaliphatic, aliphatic(aryl),heteroaliphatic(aryl), aliphatic(heteroaryl) orheteroaliphatic(heteroaryl) moiety, or an aryl or heteroaryl moiety;wherein each of the foregoing aliphatic and heteroaliphatic moieties maybe substituted or unsubstituted, cyclic or acyclic, saturated orunsaturated or linear or branched; and each of the foregoing aryl,heteroaryl, alkylaryl or alkylheteroaryl moieties may be substituted orunsubstituted; and pharmaceutically acceptable derivatives thereof. 3.The compound of claim 1, wherein R¹ is hydrogen or an alkyl,heteroalkyl, aryl or heteroaryl moiety substituted with Z, wherein Z ishydrogen, —(CH₂)_(q)OR^(Z), —(CH₂)_(q)SR^(Z), —(CH₂)_(q)N(R^(Z))₂,—(C═O)R^(Z), —(C═O)N(R^(Z))₂, or an aliphatic, heteroaliphatic, aryl,heteroaryl, -(aliphatic)aryl, -(aliphatic)heteroaryl,-(heteroaliphatic)aryl, or -(heteroaliphatic)heteroaryl moiety, whereinq is 0-4, and wherein each occurrence of R^(Z) is independentlyhydrogen, a protecting group, a solid support unit, or an aliphatic,heteroaliphatic, aryl, heteroaryl, -(aliphatic)aryl,-(aliphatic)heteroaryl, -(heteroaliphatic)aryl, or-(heteroaliphatic)heteroaryl moiety; wherein each of the foregoing alkylor heteroalkyl moieties may be substituted or unsubstituted, linear orbranched, cyclic or acyclic, saturated or unsaturated; and wherein eachof the foregoing aryl, heteroaryl, -(alkyl)aryl, -(alkyl)heteroaryl,-(heteroalkyl)aryl, or -(heteroalkyl)heteroaryl moieties may besubstituted or unsubstituted.
 4. The compound of claim 3, wherein R¹ ishydrogen, lower alkyl, a substituted or unsubstituted phenyl or -(loweralkyl)phenyl moiety, —(CH₂)_(n)OR^(z), —[(CH₂)_(n)O]_(m)R^(z),—(CH₂)_(n)—Ar—(CH₂)_(m)OR^(z); wherein n and m are each independentlyintegers from 1-6, Ar represents a substituted or unsubstituted aryl orheteroaryl moiety, and R^(z) is independently hydrogen, a protectinggroup, a solid support unit, or an aliphatic, heteroaliphatic, aryl,heteroaryl, -(aliphatic)aryl, -(aliphatic)heteroaryl,-(heteroaliphatic)aryl, or -(heteroaliphatic)heteroaryl moiety; whereineach of the foregoing alkyl or heteroalkyl moieties may be substitutedor unsubstituted, linear or branched, cyclic or acyclic, saturated orunsaturated; and wherein each of the foregoing aryl, heteroaryl,-(alkyl)aryl, -(alkyl)heteroaryl, -(heteroalkyl)aryl, or-(heteroalkyl)heteroaryl moieties may be substituted or unsubstituted.5. The compound of claim 4, wherein R¹ is hydrogen, ethyl, or has one ofthe structures:

wherein R^(z) is as defined in claim
 4. 6. The compound of claim 1,wherein R² is hydrogen or an alkyl, heteroalkyl, aryl or heteroarylmoiety substituted with Z, wherein Z is hydrogen, —(CH₂)_(q)OR^(Z),—(CH₂)_(q)SR^(Z), —(CH₂)_(q)N(R^(Z))₂, —(C═O)R^(Z), —(C═O)N(R^(Z))₂, oran aliphatic, heteroaliphatic, aryl, heteroaryl, -(aliphatic)aryl,-(aliphatic)heteroaryl, -(heteroaliphatic)aryl, or-(heteroaliphatic)heteroaryl moiety, wherein q is 0-4, and wherein eachoccurrence of R^(z) is independently hydrogen, a protecting group, asolid support unit, or an aliphatic, heteroaliphatic, aryl, heteroaryl,-(aliphatic)aryl, -(aliphatic)heteroaryl, -(heteroaliphatic)aryl, or-(heteroaliphatic)heteroaryl moiety; wherein each of the foregoing alkylor heteroalkyl moieties may be substituted or unsubstituted, linear orbranched, cyclic or acyclic, saturated or unsaturated; and wherein eachof the foregoing aryl, heteroaryl, -(alkyl)aryl, -(alkyl)heteroaryl,-(heteroalkyl)aryl, or -(heteroalkyl)heteroaryl moieties may besubstituted or unsubstituted.
 7. The compound of claim 6, wherein R² ishydrogen, lower alkyl, a substituted or unsubstituted phenyl or -(loweralkyl)phenyl moiety, —(CH₂)_(n)OR^(z), —[(CH₂)_(n)O]_(m)R^(z),—(CH₂)_(n)—Ar—(CH₂)_(m)OR^(z); wherein n and m are each independentlyintegers from 1-6, Ar represents a substituted or unsubstituted aryl orheteroaryl moiety, and R^(z) is independently hydrogen, a protectinggroup, a solid support unit, or an aliphatic, heteroaliphatic, aryl,heteroaryl, -(aliphatic)aryl, -(aliphatic)heteroaryl,-(heteroaliphatic)aryl, or -(heteroaliphatic)heteroaryl moiety; whereineach of the foregoing alkyl or heteroalkyl moieties may be substitutedor unsubstituted, linear or branched, cyclic or acyclic, saturated orunsaturated; and wherein each of the foregoing aryl, heteroaryl,-(alkyl)aryl, -(alkyl)heteroaryl, -(heteroalkyl)aryl, or-(heteroalkyl)heteroaryl moieties may be substituted or unsubstituted.8. The compound of claim 6, wherein R² is hydrogen or has one of thestructures:

wherein R^(z) is as defined in claim
 6. 9. The compound of claim 1,wherein R³ is an alkyl, heteroalkyl, heteroaryl, -(alkyl)aryl,-(alkyl)heteroaryl, -(heteroalkyl)aryl, or -(heteroalkyl)heteroarylmoiety; wherein each of the foregoing alkyl or heteroalkyl moieties maybe substituted or unsubstituted, linear or branched, cyclic or acyclic,saturated or unsaturated; and wherein each of the foregoing heteroaryl,-(alkyl)aryl, -(alkyl)heteroaryl, -(heteroalkyl)aryl, or-(heteroalkyl)heteroaryl moieties may be substituted or unsubstituted.10. The compound of claim 9, wherein R³ has one of the structures:


11. The compound of claim 1, wherein R⁴ is hydrogen or an alkylheteroalkyl, aryl, heteroaryl, -(alkyl)aryl, -(alkyl)heteroaryl,-(heteroalkyl)aryl, or -(heteroalkyl)heteroaryl moiety; wherein each ofthe foregoing alkyl or heteroalkyl moieties may be substituted orunsubstituted, linear or branched, cyclic or acyclic, saturated orunsaturated; and wherein each of the foregoing aryl, heteroaryl,-(alkyl)aryl, -(alkyl)heteroaryl, -(heteroalkyl)aryl, or-(heteroalkyl)heteroaryl moieties may be substituted or unsubstituted.12. The compound of claim 11, wherein R⁴ is hydrogen alkyl orheteroalkyl.
 13. The compound of claim 1, wherein R⁵ and R⁶ are eachindependently hydrogen or an alkyl, heteroalkyl, aryl, heteroaryl,-(alkyl)aryl, -(alkyl)heteroaryl, -(heteroalkyl)aryl, or-(heteroalkyl)heteroaryl moiety; or wherein R⁵ and R⁶, taken together,form a substituted or unsubstituted, saturated or unsaturated cyclicmoiety comprising 5-12 carbon atoms, 0-5 oxygen atoms, 0-5 sulfur atomsand 1-5 nitrogen atoms; and wherein each of the foregoing alkyl orheteroalkyl moieties may be substituted or unsubstituted, linear orbranched, cyclic or acyclic, saturated or unsaturated; and wherein eachof the foregoing aryl, heteroaryl, -(alkyl)aryl, -(alkyl)heteroaryl,-(heteroalkyl)aryl, or -(heteroalkyl)heteroaryl moieties may besubstituted or unsubstituted.
 14. The compound of claim 1, wherein—NR⁵R⁶ is one of the following the structures:


15. (canceled)
 16. (canceled)
 17. (canceled)
 18. The compound of claim 1having the structure:


19. The compound of claim 1 having the structure:


20. The compound of claim 1 having the structure:


21. The compound of claim 1 having the structure:


22. (canceled)
 23. A library of compounds comprising a plurality oflibrary members, wherein at least two library members are a compound ofclaim
 1. 24. (canceled)
 25. (canceled)
 26. The library of claim 23,wherein the library comprises at least 100 compounds.
 27. The library ofclaim 23, wherein the library comprises at least 1,000 compounds. 28.The library of claim 23, wherein the library comprises at least 2,000compounds.
 29. The library of claim 23, wherein the library comprises atleast 10,000 compounds.
 30. A pharmaceutical composition comprising acompound according to claim 1 and a pharmaceutically acceptable carrier.31-39. (canceled)